Detection of mycobacteria

ABSTRACT

The present invention is directed to methods and compositions for the detection of infection and disease due to members of the genus Mycobacterium. In particular, the present invention is well-suited to the detection and identification of patients with disease or infection due to  M. tuberculosis  or MAC.

This application was made with government support under A145244 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates to the detection and diagnosis ofinfection and disease due to the mycobacteria, especially Mycobacteriumtuberculosis and other mycobacteria commonly associated with disease inimmunocompromised patients including those with acquiredimmunodeficiency syndrome (AIDS). The present invention is suited forrapid screening of large populations for the presence of M. tuberculosiscarriers, as well as diagnosing and monitoring disease or infection inpatients who present at healthcare or public health facilities.

BACKGROUND OF THE INVENTION

Organisms within the genus Mycobacterium include obligate parasites,saprophytes, and opportunistic pathogens. Most species are free-livingin soil and water, but for species such as M. tuberculosis and M.leprae, the causative agents of tuberculosis and leprosy respectively,the major ecological niche is the tissue of humans and otherwarm-blooded animals.

Despite the fact that most mycobacteria do not cause disease, arelatively small group of organisms within the genus is responsible fora large percentage of human morbidity and mortality worldwide.Tuberculosis remains a major global health problem, with nearly onethird of the world's population infected. Indeed, tuberculosis is theleading cause of death due to a single infectious agent. In addition.the World Health Organization estimates that worldwide, there are 8-10million new cases and over 3 million deaths directly attributed to thisdisease reported worldwide (A. Kochi, The global tuberculosis situationand the new control strategy of the World Health Organization, Tubercle72:1-6 [1991]).

M. tuberculosis is exceptionally easily transmitted, as it is carried inairborne particles termed “droplet nuclei,” produced when a patient withactive tuberculosis coughs. These particles are from 1-5μ in size, andare readily suspended in air currents. Infection occurs when dropletnuclei are inhaled and reach the terminal airways of the new host'slungs. Usually, the host immune response limits the multiplication andspread of the organism, although some organisms may remain dormant, butviable, for many years post-infection. Individuals infected with M.tuberculosis but without disease, usually have a positive skin test(i.e., with purified protein derivative [PPD]), but are asymptomatic andgenerally not infectious. However, latently infected individuals have a10% risk for developing active tuberculosis at some point during theirlife; the risk is greatest within the first two years post-infection.For HIV-positive individuals, the risk is much greater, with the risk at10-15% per year for progression to active disease (F. S. Nolte and B.Metchock, “Mycobacterium,” in Manual of Clinical Microbiology, SixthEdition, ASM Press: Washington, [1995],pp. 400-437).

Co-infection with human immunodeficiency virus (HIV) and M. tuberculosishas resulted in staggering increases in tuberculosis rates—as much as200% in the past 4 years, particularly in impoverished countries withfew resources available to control this epidemic. Yet even westernindustrialized countries have reported increases in tuberculosis ratesof from 2 to 14% per year during the past decade (World HealthOrganization TB Programme, quoted in “TB: A Global Emergency,” WHO,1994). These increases, coupled with the emergence ofmulti-drug-resistant strains, and the recognition that tuberculosis isone HIV-related opportunistic infection which can be readily transmittedto HIV-uninfected persons, have focused the attention of physicians,researchers, and public health workers on issues related to tuberculosiscontrol, particularly in terms of development of improved vaccines fortuberculosis prevention and improved tests for tuberculosis diagnosis.

In the United States, aggressive approaches to tuberculosis controlincluding isolation of patients in facilities such as sanitoria and thedevelopment of drugs effective against M. tuberculosis resulted in asteady decline in the incidence of tuberculosis until about 1985, whenthe trend reversed and the reports of new tuberculosis cases began toincrease. If the trend for the years 1980-1984 is used to calculate thenumber of expected cases, the Centers for Disease Control and Prevention(CDC) estimated that between 1985-1992, approximately 51,000 excesscases have accumulated (D. E. Snider et al., “Global burden oftuberculosis,” in B. R. Bloom (ed.), Tuberculosis: Pathogenesis,Protection and Control, American Society for Microbiology, Washington,D.C., [1994], pp. 3-11).

A number of contributory factors are likely to be responsible for theobserved increase in tuberculosis cases, including the AIDS pandemic,immigration from areas with high endemicity of tuberculosis, generaldeterioration of the health care infrastructure, transmission inhigh-risk environments (e.g., homeless shelters), and the increase inthe number of multi-drug resistant M. tuberculosis strains (Nolte andMetchock, supra, at p. 400). Unless the effectiveness and availabilityof methods and drugs to detect and treat tuberculosis do notsubstantially improve, it is expected that over 30 million deaths and 90million new cases of tuberculosis will occur in the years between1990-2000 (Snider et al., at p. 10).

Although it is the major cause, organisms other than M. tuberculosis aresometimes associated with tuberculosis in humans and other animals.These organisms are included in the “M. tuberculosis complex,” whichincludes M. bovis, M. africanum, and M. microti, as well as M.tuberculosis. M. bovis causes tuberculosis in cattle, humans and otherprimates, carnivores (e.g., dogs and cats), swine, parrots, and somebirds of prey. Human disease is virtually indistinguishable from thatcaused by M. tuberculosis, and is treated in a similar manner (Nolte andMetchock, supra, at 402). Similarities between M. bovis and M.tuberculosis led to the development of the bacillus of Calmette-Guérin(BCG) an attenuated form of M. bovis, as a vaccine against tuberculosisin many parts of the world (See e.g., W. K. Joklik et al. (eds.),Zinsser Microbiology, 18th ed., Appleton-Century Crofts, Norwalk, Conn.,[1984], p. 564).The human health problems associated with M. bovis werelargely responsible for the development of methods for thepasteurization of milk and the adoption of compulsory pasteurization inthe early 1900s (See, C. O. Thoen, “Tuberculosis in wild and domesticmammals,” in B. R. Bloom (ed.) Tuberculosis: Pathogenesis, Protectionand Control, American Society for Microbiology, Washington, D.C. [1994],pages 157-162). M. africanum has been reported from cases oftuberculosis in tropical Africa. M. microti causes generalizedtuberculosis in voles, and produces local lesions in such animals asguinea pigs, rabbits, and calves (Nolte and Metchock, supra, at 402).

Thus, M. tuberculosis is not the only respiratory pathogen of greatpublic health concern, and neither is the M. tuberculosis complex.Recent developments in the taxonomy and study of the mycobacteria haveresulted in recognition of M. avium complex (MAC) organisms as the causeof disseminated disease in immunocompromised patients, in particularAIDS patients. The two major species associated with MAC are M. aviumand M. intracellulare. However, the MAC includes 28 serovars of thesetwo distinct species, although three additional serovars of M.scrofulaceum (i.e., M. avium—M. intracellulare—M. scrofulaceum complex)were previously included. Within the M. avium species, three subspecieshave been proposed, based on phenotypic and genotypic characteristics(M. avium subspecies avium, M. avium subspecies paratuberculosis, and M.avium subspecies silvaticum) (M.-F. Thorel et al., Numerical taxonomy ofmycobactin-dependent mycobacteria, emended description of Mycobacteriumavium, and description of Mycobacterium avium subsp. avium, subsp. nov.M. avium subsp. paratuberculosis, subsp. nov., and Mycobacterium aviumsubsp. silvaticum subsp. nov., Int. J. Syst. Bacteriol., 40:254-260[1990]). As additional information is gathered on the geneticrelationships among the mycobacteria, it is likely that changes willoccur in the taxonomy of these organisms.

M. avium, an important pathogen of poultry and swine was recognized as acause of disease in chickens in the late 1800s, but was not recognizedas a cause of human disease until 1943 (See, Nolte and Metchock, supra,at 402). M. intracellulare is usually associated with disease in swineand cattle. In addition to its veterinary significance, MAC is animportant pathogen of immunocompromised patients, especially those withAIDS. Indeed, disseminated MAC infection is the most commonopportunistic infection observed late in the course of HIV disease. Ithas been reported that the frequency of disease due to MAC rises from 3%per year for individuals with CD4 counts of 100-200/μl to 39% at CD4counts of <10/μl (S. D. Nightingale et al., “Incidence of M. aviumintracellulare complex bacteraemia in HIV-positive patients,” J. Infect.Dis., 165:108-25 [1992]). Disease with MAC is characterized by fever,cachexia, hepatic dysfunction, and anemia. As with infection with M.tuberculosis, infection with MAC may promote HIV expression, leading toaccelerated HIV disease progression.

Human acquisition of M. avium appears to occur via inhalation oringestion of fresh water in which bacilli are concentrated (K. L. Fry etal., “Epidemiology of infection by nontuberculous mycobacteria. VI.Identification and use of epidemiologic markers for studies ofMycobacterium avium, M. intracellulare, and M. scrofulaceum,” Am. Rev.Respir. Dis., 134:39-43[1986]). It is hypothesized that local infectionis followed by hematogenous spread to organs of the reticuloendothelialsystem. It is here, in bone marrow and lymph nodes, that the number ofMAC colony forming units (CFU) ultimately rise to levels several logshigher than are present in blood (D. Peterson et al., “M. avium complex(MAC) disease in HIV-infected patients is a uniform infection of bonemarrow that does not correlate with the level of infection in blood,”Natl. Conf. Hum. Retrovir. Rel. Pathogens 2:56 [1995]). The observedmycobacteremia apparently represents “spillover” of bacilli from theseheavily infected organs. Alternatively, MAC mycobacteremia may occurintermittently, arising from the gastrointestinal tract, withoutinevitably causing tissue infection.

Recently, our understanding of the mycobacteria, including the recentlydescribed species (e.g., M. genavense) associated with disseminateddisease in HIV-infected individuals, as well as the potential pathogensM. asiaticum, M. haemophilum, M. malmoense, M. shimoidei, and M.celatum, has greatly increased, largely due to an increased interest inopportunistic pathogens, especially those associated with disease inAIDS patients. Nonetheless, many problems remain unresolved, andreliable, rapid methods are needed for the detection of latent andactive mycobacterial infections, especially in the case of AIDSpatients.

Treatment of Mycobacterial Disease and Infection

The unique properties of the mycobacterial cell wall, growth rates, andother factors have provided avenues as well as detours in ourdevelopment of methods for detection and treatment of mycobacterialdisease. The mycobacteria characteristically have cell walls with a highlipid content, including waxes such as mycolic acid. The properties ofthis waxy cell wall provide the “acid-fast” nature of the organisms, asonce dye is taken into the cells, they are not easily decolorized, evenwith acid-alcohol. Thus, unlike most organisms, the mycobacteria aresaid to be “acid fast” and are often referred to as “acid-fast bacilli”or “AFB.”

The growth rate of the mycobacteria ranges from slow to very slow, withgeneration times ranging from two to 24 hours. Most isolates, includingM. tuberculosis require long incubation periods (i.e., 4-8 weeks fortraditional culture methods) under optimal conditions for growth to beeasily visible in vitro. Once the organisms from a primary culture havegrown, biochemical and other tests must be done in order to provide anidentification. This is an unacceptably long time between sampling and adefinitive identification of the organism causing disease in a patient.

The slow growth rate and the pathogenic processes of M. tuberculosiscontribute to problems encountered in treating tuberculosis patients. Asmost antimicrobial drugs work against actively growing cultures, therelatively metabolically inactive mycobacteria enclosed withinrelatively impermeable waxy cell walls are generally unaffected by mostdrugs commonly used to combat bacterial disease.

The first line of drugs used against M. tuberculosis include isoniazid(INH), rifampin, pyrazinamide, ethambutol, and streptomycin. The secondline drugs include para-amino salicylic acid, ethionamide, cycloserine,capromycin, kanamycin, amikacin, ciprofloxacin, ofloxacin and rifabutin.It is recommended that patients initially be treated with INH, rifampin,ethambutol, and pyrazinamide for 2 months. Those patients with fullydrug-susceptible isolates then may be treated with INH and rifampin foran additional four months (American Thoracic Society and Centers forDisease Control, “Treatment of tuberculosis and adults and children,”Am. Rev. Respir. Dis., 134:355-363 [1986]). Patients with isolatesresistant to either INH or rifampin must be treated with alternativeregimens for longer duration. In all cases, successful treatment ofpatients must continue long after acid-fast bacilli are no longerdetected in sputum samples.

Drug resistant M. tuberculosis strains of have become a serious concernworldwide. In a recent nationwide survey of drug resistance amongtuberculosis cases reported during the first quarter of 1991, it wasfound that 14.9% had isolates that were resistant to at least oneanti-tuberculosis drug, and 3.3% had isolates resistant to both INH andrifampin (Centers for Disease Control, “National MDR-TB Task Force,national action plan to combat multidrug-resistant tuberculosis,”Morbid. Mortal. Wkly. Rept. 41:1-48 [1992]). This is of grave concern,as INH and rifampin are the most effective drugs in our arsenal againstM. tuberculosis.

For MAC, the concerns are potentially even more significant. Strains ofMAC have been reported to be intrinsically resistant toanti-tuberculosis drugs and many other antimicrobials due to failure ofthese drugs to penetrate the lipid-rich cell wall (N. Rastogi et al.,“Enhancement of drug susceptibility of Mycobacterium avium by inhibitorsof cell envelope synthesis,” Antimicrob. Agents Chemother., 34:759-764[1990]; and N. Rastogi et al., “Simplified acetylcysteine-alkalidigestion-decontamination procedure for isolation of mycobacteria fromclinical specimens,” J. Clin. Microbiol., 25:1428-1438 [1987]). Indeed,optimal regimens for treatment of either chronic pulmonary disease ordisseminated MAC infections in AIDS patients have not been defined. Inaddition, no therapeutic regimen has been shown to be of sustainedclinical benefit for patients with disseminated MAC (Nolte and Metchock,supra, at p. 428). It is recommended that patients with HIV and <100CD4⁺ cells be given prophylaxis against MAC that is to be continued forthe patient's life, unless multi-drug therapy becomes necessary due todisseminated disease (Centers for Disease Control, “Recommendations onprophylaxis and therapy for disseminated Mycobacterium avium complex foradults and adolescents infected with human immunodeficiency virus,”Morbid. Mortal. Wkly Rept., 42(RR):14-20 [1993]). Preventive therapy forMAC can be problematic because of interactions with other drugs commonlyused in HIV infection, particularly the new protease inhibitors.Although no optimal treatment regimen has been defined for disseminatedMAC, the U.S. Public Health Service Task Force on Prophylaxis andTherapy for MAC recommends that treatment continued for the lifetime ofthe patient, even if improvement is noted; this treatment should includeat least two chemotherapeutic agents, one of which should beazithromycin or clarithromycin.

The situation is even potentially more grim if other Mycobacteriumspecies are associated with disease in a patient, as almost all of thestrains of the “rapid grower” (ie., in vitro growth may be observed inas few a two days) Mycobacterium species (i.e., M. chelonae, M.fortuitum, M. abscessus, etc.) are resistant to the anti-tuberculosisdrugs. Thus, prophylactic treatment of immunocompromised patients isproblematic and treatment is dependent upon the results of antimicrobialsusceptibility testing of patient isolates.

Detection of Mycobacterial Disease and Infection

The field of diagnostic and clinical microbiology has continued toevolve, and yet, there remains a general need for systems that providerapid and reliable detection of disease and infection due tomicroorganisms such as M. tuberculosis. Nontheless, the role of theclinical mycobacteriology laboratories cannot be underestimated in viewof their potential contributions in controlling the spread oftuberculosis and non-tuberculosis disease through the timely detection,isolation, identification, and determination of drug susceptibility ofthese organisms. There is a “new sense of urgency” regarding thereporting of acid-fast smear, cultures, and drug susceptibility resultsto physicians, prompted in large part to the emergence of multi-drugresistant strains of M. tuberculosis (Nolte and Metchock, supra, at p.400). Traditionally, tuberculosis surveillance has involved the use ofpreliminary skin tests (e.g., tuberculin tests), with positives beingfurther evaluated for active disease by radiographic analysis (i.e.,chest X-rays), and sputum cultures. Other samples are sometimessubmitted to the laboratory for culture, including blood,bronchoalveolar lavage fluid, bronchial washings, gastric lavage fluids,urine, body fluids (e.g., cerebrospinal [CSF], pleural, peritoneal,pericardial, etc.), tissues (e.g., lymph nodes, skin, or other biopsymaterials), abscess contents, aspirated fluids, skin lesions, andwounds.

In contrast to the situation with tuberculosis, blood cultures are oftenused for the isolation of MAC from immunocompromised patients,especially those with AIDS. Positive MAC blood cultures are generally,but not always, associated with clinical evidence of tissue infection,which typically can involve the bone marrow, liver, or lymph nodes (J.Havlik et al., “Disseminated Mycobacterium avium complex infection:clinical identification and epidemiologic trends,” J. Infect. Dis.,165:577-580 [1992], F J. Torriani et al., “Autopsy findings in AIDSpatients with M. avium complex bacteremia,” J. Infect. Dis., 170:1601-5[1994]). Thus, detection of MAC in tissue samples provides usefulinformation on a patient's status. However, culturing of blood remainsthe most commonly used method for diagnosis of MAC infection, primarilybecause of the requirement for an invasive procedure to sample infectedtissues.

Culture Methods. Once a specimen has been received in the laboratorysuspected of containing mycobacteria, the specimen will generally bestained and examined for the presence of AFB. Sputum and other “dirty”specimens are decontaminated and concentrated prior to staining andculturing. Specimens are inoculated onto either solid egg-based media(i.e., Lowenstein-Jensen agar), or liquid medium, in which growth ismore rapid. Various commercial growth media systems and methods areavailable for detection of mycobacteria, including BACTEC(Becton-Dickinson Diagnostic Instrument Systems), Septi-Chek(Becton-Dickinson Microbiology Systems), and the Isolator system(Wampole). Automated detection methods (e.g., those based on productionof radiolabelled CO₂, turbidity or light) have been developed toidentify cultures with microbial growth. However, growth of M.tuberculosis and other mycobacterial species must be confirmed by othermethods. Methods presently accepted for detection and identification ofare described in great detail in various publications (see e.g., N.Master (section editor) Section 3. “Mycobacteriology,” in H. E. Isenberg(editor in chief), Clinical Microbiology Procedures Handbook, volume 1,3.0.1-3.16.4 [1994]).

Immunological Methods. Methods for diagnosis of tuberculosis based onimmunologic methods such as detection of delayed type hypersensitivity(DTH) skin responses, as well as the detection of anti-mycobacteriaantibodies, or mycobacterial antigens have been studied. Historically,skin tests have been commonly used as indicators of infection with M.tuberculosis. The tuberculin skin test, still commonly in use, was thefirst immunodiagnostic test developed for detection of tuberculosis.Problems with this test include its inability to distinguish activedisease from past sensitization, as well as its unknown predictiveaccuracy (D. Snider, “The tuberculin skin test,” Am. Rev. Resp. Dis.,125(Suppl.):108-118 [1982]). Even among healthy skin test reactors, thetest cannot distinguish those individuals with continued latentinfection (in whom there is a continued risk of developing activedisease) from those in whom a protective immune response has eradicatedthat infection. In vitro tests to determine the cell-mediated responsesto mycobacterial antigens have also been described. However, they areexpensive, technically demanding to perform and interpret, and provideno additional data than are available from skin testing (See e.g., Nolteand Metchock, supra, at p. 426).

Despite the fact that much effort has been devoted to development ofserological tests for tuberculosis, these methods have not foundwidespread clinical use (E. Bardana, “Universal occurrence of antibodiesto tubercle bacilli in sera from non-tuberculous and tuberculousindividuals,” Clin. Exp. Immunol., 13:65-77 [1973]; and T. M. Daniel andS. M. Debanne, “The serodiagnosis of tuberculosis and othermycobacterial disease by enzyme-linked immunosorbent assay,” Am. Rev.Resp. Dis., 158:678-680 [1987]). Nonetheless, perhaps because antibodydetection methods are commonly used in the diagnosis of infectiousdisease, assay methods based on anti-mycobacterial antibody detectionhave been investigated.

Antibody Detection. Several studies have focused on methods to determinethe anti-mycobacterial antibody level in patients' sera. These studieshave used a variety of antigen preparations, including crude extracts,purified native antigens, and recombinant proteins. Immunoassays,including ELISAs and radioimmunoassays (RIA) have been used in many ofthese studies (See e.g., E. G. Wilkins et al., “A Rapid, Simple EnzymeImmunoassay for Detection of Antibody to Individual Epitopes in theSerodiagnosis of Tuberculosis,” Eur. J. Clin. Microbiol. Infect. Dis.,10: 559-563 [1991]; R. G. Benjamin et aL, “Serodiagnosis of TuberculosisUsing the Enzyme-Linked Immunoabsorbent Assay (ELISA) of Antibody toMycobacterium tuberculosis Antigen 5¹⁻³,” Amer. Rev. Respir. Dis.,126:1013-1016 [1982]); R. Maes et al., “Development of an EnzymeImmunoassay for the Serodiagnostic of Tuberculosis and Mycobacterioses,”Med. Microbiol. Immunol., 178: 323-335 [1989]); S. B. Kalish et al.,“Use of an Enzyme-Linked Immunosorbent Assay Technique in theDifferential Diagnosis of Active Pulmonary Tuberculosis in Humans,” J.Infect. Dis., 147: 523-530 [1983]); E. Nassau et al., “The Detection ofAntibodies to Mycobacterium tuberculosis by Microplate Enzyme-LinkedImmunosorbent Assay (ELISA),” Tubercle, 57: 67-70 [1976]; F. L.Garcia-Carreno, “Enzyme Immunoassay Using BCG in Serodiagnosis ofPulmonary Tuberculosis,” J. Hyg., 97: 483-487 [1986]; R. Hernandez etal., “Sensitive Enzyme Immunoassay for Early Diagnosis of TuberculousMeningitis,” J. Clin. Microbiol., 20:533-535 [1984]; J. A. McDonough etal., “Microplate and Dot Immunoassays for the Serodiagnosis ofTuberculosis,” J. Lab Clin. Med., 120:318-322 [1992]; E. Sada et al.,“An ELISA for the Serodiagnosis of Tuberculosis Using a 30,00-Da NativeAntigen of Mycobacterium tuberculosis,” J. Infect. Dis., 162:928-931[1990]; A. Mathai et al., “Rapid Diagnosis of Tuberculous Meningitiswith a Dot Enzyme Immunoassay to Detect Antibody in CerebrospinalFluid,” Eur. J. Clin. Microbiol. Infect. Dis., 10:440-443 [1991]; M.Turneer et al., “Humoral Immune Response in Human Tuberculosis:Immunoglobulins G, A, and M Directed against the Purified P32 ProteinAntigen of Mycobacterium bovis Bacillus Calmette-Guerin,” J. Clin.Microbiol., 26: 1714-1719 [1988]; N. K. Kaushik et al., “SerodiagnosticEfficiency of Phospholipid Associated Protein of Mycobacteriumtuberculosis H₃₇Rv,” Med. Microbiol. Immunol., 182:317-327 [1993]; D.Kumar et al., “Identification of a 25-Kilodalton Protein ofMycobacterium bovis BCG to Distinguish BCG Strains from Mycobacteriumtuberculosis,” J. Clin. Microbiol., 34: 224-226 [1996]; Chandramuki etal. “Levels of Antibody to Defined Antigens of Mycobacteriumtuberculosis in Tuberculous Meningitis,” J. Clin. Microbiol., 27:821-825 [1989]; K. A. Near et al., “Use of Serum Antibody and LysozymeLevels for Diagnosis of Leprosy and Tuberculosis,” J. Clin. Microbiol.,30: 1105-1110 [1992]; and H. Miöner et al., “Diagnosis of TuberculousMeningitis: A Comparative Analysis of 3 Immunoassays, An Immune ComplexAssay and the Polymerase Chain Reaction,” Tubercle Lung Dis., 76:381-386 [1995].

Although as listed above, numerous researchers have attempted to developimmunodiagnostic systems based on detection of antibody directed againstmycobacterial antigens, no antibody tests have been accepted orsufficiently developed for routine diagnosis of tuberculosis. This is inlarge part due to the fact that the specificities of tests that usecrude antigens are too low to be useful clinically. In addition, not allpatients respond to the same mycobacterial antigens; any increasedspecificity achieved by using purified antigens is compromised by aconcomitant decrease in sensitivity (See e.g., P. S. Jackett et al.,“Specificity of Antibodies to Immunodominant Mycobacterial Antigens inPulmonary Tuberculosis,” J. Clin.Microbiol., 26: 2313-2318 [1988]).Lastly, immune responses are inadequate in immunocompromised hosts whoare at greatest risk of developing tuberculosis. In order to avoid theproblems associated with detecting host immune response, detectionmethods for mycobacterial antigens themselves have been investigated.

Antigen Detection. Detection of microbial antigens in fluids remote fromthe site of infection has been applied to diagnosis and monitoring oftherapy for several infectious diseases other than tuberculosis,including cryptococcosis, histoplasmosis, and, on an experimental basis,leprosy. The type of antigen and the optimal strategy for testing variesaccording to the illness. In the case of cryptocococcis, apolysaccharide capsular antigen can be detected in cerebrospinal fluidand blood using a simple latex agglutination test (A. A. Gal et al.,“The clinical laboratory evaluation of cryptococcal infections in theacquired immunodeficiency syndrome,” Diagn. Microbiol. Infect. Dis.,7:249-54 [1987]). In disseminated histoplasmosis, a heat stablepolysaccharide antigen may be detected in blood, CSF, and urine byradioimmunoassay (L. J. Wheat et al., “Diagnosis of disseminatedhistoplasmosis by detection of Histoplasma capsulatum antigen in serumand urine specimens,” N. Engl. J. Med., 314:83-8 [1986]). In leprosy,serum levels of M. leprae phenolic glycolipid I correlate with bacillaryload at diagnosis and during therapy, ranging from 12 ng/ml inpaucibacillary patients to as high as 8000 ng/ml in multibacillarypatients (D. B. Young et al., “Detection of phenolic glycolipid I insera from patients with lepromatous leprosy,” J. Infect. Dis.,152:1078-81 [1985]). However, the successes with these organisms havenot been mirrored in diagnosis of tuberculosis and MAC disease, with theexception of one report for MAC, described below.

Detection of Mycobacterial Antigens. Previous reports of antigendetection assays for rapid diagnosis of tuberculosis have been limitedto examination of fluids obtained from the site of clinical disease,such as cerebrospinal fluid, sputum, or bronchoalveolar lavage fluid (E.Sada et al., “Detection of mycobacterial antigens in cerebrospinal fluidof patients with tuberculous meningitis by enzyme-linked immunosorbentassay,” Lancet 2:651-2 [1983]; I. O. al Orainey et al., “Detection ofmycobacterial antigens in sputum by an enzyme immunoassay,” Eur. J.Clin. Microbiol. Infect. Dis., 11:58-61 [1992]; R. Kansal et al.,“Detection of mannophosphoinositide antigens in sputum of tuberculosispatients by dot enzyme immunoassay,” Med. Microbiol. Immunol. Berl.,180:73-8 [1991]; M. A. Yanez et al., “Determination of mycobacterialantigens in sputum by enzyme immunoassay,” J. Clin. Microbiol., 23:822-5[1986]; G. V. Kadival et al., “Radioimmunoassay of tuberculous antigen,”Indian J. Med. Res., 75:765-70 [1982], A. Chandramuki et al., “Detectionof mycobacterial antigen and antibodies in the cerebrospinal fluid ofpatients with tuberculous meningitis,” J. Med. Microbiol., 20: 239-247[1985]; C. L. Cambiaso et al., “Immunological detection of mycobacterialantigens in infected fluids, cells and tissues by latexagglutination—Animal model and clinical application,” J. Immunol. Meth.,129: 9-14 [1990]). A number of other antigen assays have also beendescribed (See e.g., T. M. Daniel, “Rapid diagnosis of tuberculosis:Laboratory techniques applicable in developing countries,” Rev. Infect.Dis., 2(Supplement 2): S471-S478 ([1989]).

In other studies, the infected fluids were placed in liquid culture fora short period, and mycobacterial products were detected in the culturemedium (A. Raja et al., “Specific detection of Mycobacteriumtuberculosis in radiometric cultures by using an immunoassay for antigen5,” J. Infect. Dis., 158:468-70 [1988]; A. Raja et al., “The detectionby immunoassay of antibody to mycobacterial antigens and mycobacterialantigens in bronchoalveolar lavage fluid from patients with tuberculosisand control subjects,” Chest 94: 133-137 [1988]; R. Schoningh R et al.,“Enzyme immunoassay for identification of heat-killed mycobacteriabelonging to the Mycobacterium tuberculosis and Mycobacterium aviumcomplexes and derived from early cultures,” J. Clin. Microbiol.,28:708-13 [1990]; A. Drowart et al., “Detection of mycobacterialantigens present in short-term culture media using particle countingimmunoassay,” Am. Rev. Respir. Dis., 147:1401-6 [1993]). The detectionthreshold of these assays ranged from 1 ng to 1 μg/ml. All used apolyclonal antiserum rather than monoclonal antibody for capture;several used the same serum for both capture and detection. In none ofthese reports were fluids remote from the site of infection studied, andin none could the assay identify subjects with latent infection.

M. tuberculosis Antigens. In contrast to the situation with manybacteria, the antigens of M. tuberculosis are a remarkably complexmixture of proteins, polysaccharides, and lipids. The polysaccharideantigens share antigenic cross-reactivity with Nocardia, thecorynebacteria, and staphylococci, and generally do not elicit delayedtype hypersensitivity (DTH) (Y. Yamamura et al., “Biology of themycobacterioses. Chemical and immunological studies on peptides andpolysaccharides from tubercle bacilli,” Ann. NY Acad. Sci., 154:88-97[1968]; and S. D. Chaparas et al., “Comparison of lymphocytetransformation, inhibition of macrophage migration and skin tests usingdialyzable and nondialyzable tuberculin fractions from Mycobacteriumbovis (BCG),” J. Immunol., 107:149-53 [1971]). However, the proteinantigens of the Mycobacterium elicit a DTH response, and stimulatelymphocyte blastogenic responses in both sensitized humans and guineapigs (L. F. Affronti et al., “Some early investigations of Mycobacteriumtuberculosis,” Am. Rev. Respir. Dis., 92:1-8 [1995]); T. M. Daniel etal., “Reactivity of purified proteins and polysaccharides fromMycobacterium tuberculosis in delayed skin test and cultured lymphocytemitogenesis assays,” Infect. Immun., 9:44-7 [1974]; and S. D. Chaparaset al., “Tuberculin-active carbohydrate that induces inhibition ofmacrophage migration but not lymphocyte transformation,” Science170:637-9 [1970]).

The antigenic repertoire of M. tuberculosis includes some proteins whichare also found intracellularly, and some which appear uniquely assecreted proteins. As the technology to define these antigens hasimproved, their number has grown. In 1971, 11 antigens could beidentified by immunoprecipitation in culture filtrate (the spent mediumof cultures of M. tuberculosis after the bacilli have been removed byfiltration (B. W. Janicki et al., “A reference system for antigens ofMycobacterium tuberculosis,” Am. Rev. Respir. Dis., 104:602-4 [1971]).Antigen 5 (a 38 kD protein), and antigen 6 (a 30-32 kD protein latertermied alpha antigen, BCG 85B, and MPT59), were prominent, and appearedto have some antigenic determinants which were restricted to M.tuberculosis (T. M. Daniel et al., “Immunobiology and speciesdistribution of Mycobacterium tuberculosis antigen 5,” Infect. Immun.,24:77-82 [1979]; T. M. Daniel et al., “Demonstration of a shared epitopeamong mycobacterial antigens using a monoclonal antibody,” Clin. Exp.Immunol., 60:249-58 [1985]; and T. M. Daniel et al., “Specificity ofMycobacterium tuberculosis antigen 5 determined with mouse monoclonalantibodies,” Infect. Immun., 45:52-5 [1984]). A decade later, Closs andcolleagues identified as many as 50 distinct antigens using a system ofcrossed immunoelectrophoresis (O. Closs et al., “The antigens ofMycobacterium bovis, strain BCG, studied by crossedimmunoelectrophoresis: a reference system,” Scand. J. Immunol.,12:249-63 [1980]). The use of 2-D gel electrophoresis has increased thisnumber to greater than 100. This growing number of potential antigenshas presented a challenge in the development of diagnostic and treatmentsystems.

MAC Antigens. In contrast to these reports in which only infected fluidswere studied, a method was reported for detection of a MAC proteinantigen in urine of AIDS patients with disseminated MAC infection (A. A.Sippola et al., “Mycobacterium avium antigenuria in patients with AIDSand disseminated M. avium disease,” J. Infect. Dis., 168:466-8 [1993]).This assay utilized a goat antiserum (“K-II”) which had been developedby immunization with M. intracellulare, and which primarily recognized a22.5 kD antigen of M. intracellulare, M. avium and M. scrofulaceum, butnot M. tuberculosis. Antigenuria was detected in clinical specimensusing an assay in which supported nitrocellulose strips were dipped intovoided urine samples, which were then probed with the antiserum.Antigenuria was detected in 7/11 patients with M. avium-complex disease,and in 16/100 HIV+ controls. Two of the apparent false positive controlswere subsequently found to have disseminated M. avium infection.

Although this assay format was initially appealing in terms of itssimplicity, it has several major shortcomings, including: the use ofsupported nitrocellulose strips hinders uniform washing of multiplesamples; the binding of the target antigen to the paper is non-specific;the assay cannot be used to detect non-protein antigens, as theirbinding to the paper may not be adequate. Also, the specificity of theassay is entirely determined by that of the antiserum. It is thereforecritical that the serum not cross-react with human proteins or antigensof other pathogens, an unrealistic expectation for this assay.Therefore, the advantages of these researchers' assay may be outweighedby its disadvantages and/or be inadequate to offer clinicians asubstantial advantage when compared to the use of blood cultures fordetection of mycobacteria.

Effect of HIV Infection on Diagnosis of Mycobacterial Disease

HIV infection alters the manifestations of tuberculosis, particularly inthose patients with advanced HIV disease, in whom half havemycobacteremia and more than 75% have lymph node, hepatic, or bonemarrow involvement, particularly those with low CD4 counts (B. E. Joneset al., “Relationship of the manifestations of tuberculosis to CD4 cellcounts in patients with human immunodeficiency virus infection,” Am.Rev. Respir. Dis., 148:1292-1297 [1993]; G. I. Santos et al., “Liverdisease in patients with human immunodeficiency virus infection. Studyof 100 biopsies,” Rev. Clin. Esp., 193:115-8 [1993]; A. D. Pithie etal., “Fine-needle extrathoracic lymph-node aspiration in HIV-associatedsputum-negative tuberculosis,” Lancet 340:1504-5 [1992]; and P. F.Barnes et al., “Tuberculosis in the 1990s,” Ann. Intern. Med.,119:400-10 [1993].

However, HIV infection is accompanied by less radiographic evidence ofpulmonary disease, fewer lung zones with cavitation, and reduced numbersof mycobacterial colony-forming units in sputum (R. J. Brindle et a.,“Quantitative bacillary response to treatment in HIV-associatedpulmonary tuberculosis,” Am. Rev. Respir. Dis., 147:958-61 [1993]).Thus, the likelihood of diagnosis based on expectorated sputum isreduced in HIV-associated tuberculosis, even though the totalmycobacterial burden may be greater. This has led to the necessity ofincreased evaluation of specimens other than sputum, particularlytissues such as pleura, liver, and bone marrow. Patients with miliary ordisseminated tuberculosis, particularly those with HIV infection, oftenrequire multiple biopsies prior to initiation of therapy.

Again, despite the large number of studies, no antigen detection methodhas been developed to date which provides reliable, highly specific, andhighly sensitive results, especially for “dirty” samples such as sputum(See e.g., J. M. Grange, “The rapid diagnosis of paucibacillarytuberculosis,” Tubercle, 70:1-4 [1989]; and Nolte and Metchock, supra,at p. 426).

Importantly, serodiagnostic tests for tuberculosis, although potentiallysimple and inexpensive, have been especially hampered by poorsensitivity in HIV-infected persons in whom antibody responses arediminished (T. M. Daniel et al., “Reduced sensitivity of tuberculosisserodiagnosis in patients with AIDS in Uganda,” Tuber. Lung Dis.,75:33-7 [1994]). This is important because the patient populations thatare dually infected with HIV and M. tuberculosis is at greatest risk fordeveloping active tuberculosis (M. E. Villarino et al., “Management ofpersons exposed to multidrug-resistant tuberculosis,” Morb. Mort. Wkly.Rep., 41(RR-11):61-71 [1992]).

Diagnosis of Latent Infection with M. tuberculosis

Identification of individuals with latent M. tuberculosis infection is aproblem which cannot be addressed by current methods. The naturalhistory of M. tuberculosis infection is such that only 5-10% ofindividuals who are otherwise healthy will ever develop tuberculosis. Asmentioned above, the risk of tuberculosis is highest in the first yearfollowing infection, but tuberculosis can occur as long as 50 yearslater. Thus, the infection clearly is contained but not eradicated inmany infected but healthy individuals. Presently, there is no reliablemethod to distinguish between individuals who are latently infected andthose whose infections have been eradicated by a protective immuneresponse. Both groups have positive skin tests with purified proteinderivative (PPD), as the longevity of a positive skin test can reflectimmunologic memory as well as persistent latent infection.

The presence of immunosuppressive concurrent illnesses increases thelikelihood of recrudescent disease in tuberculosis-infected persons.This is most strongly expressed in HIV-co-infected persons, in whom therisk of re-activation of tuberculosis may be increased from 10 to 100fold. Indeed, HIV has emerged as the most significant risk factor forthe progression of latent tuberculosis to active disease (Snider et al.,at p. 5; Selwyn et al., “A prospective study of the risk of tuberculosisamong intravenous drug abusers with human immunodeficiency virusinfection,” N. Eng. J. Med., 320:545-550 [1989]). However, skin testingbecomes increasingly unreliable as an indicator of M. tuberculosisinfection as the CD4 count declines. It thus remains difficult toefficiently target those individuals who would benefit most frompreventive therapy.

Yet, identification of latently-infected persons is desirable forinitiation of tuberculosis preventive therapy. Although such therapy(daily doses of INH for nine months) can be administered to all skintest positive individuals, such therapy obviously would only benefitthose subjects who ultimately would have developed tuberculosis. Thus atleast 10-20 subjects must be treated to prevent one case oftuberculosis. The other 9-19 treated subjects are subjected to risksassociated with INH (e.g., hepatitis, neuropathy) without any potentialbenefit. This problem is compounded by the present inability to monitorthe effectiveness of preventive therapy. Some individuals will failpreventive therapy because of poor compliance with longtermadministration of INH. Others will fail because they were infected withan INH-resistant isolate. At present, there is no method to determinewhether preventive therapy has been successful in completely eradicatinglatent infection with M. tuberculosis.

In sum, there is no entirely satisfactory method for diagnosis eitheractive tuberculosis or latent mycobacterial infection. Directexamination of sputum or infected tissues is insufficiently sensitive(See e.g., L. B. Heifets and R. C. Good, “Current laboratory methods forthe diagnosis of tuberculosis,” in B. R. Bloom (ed.) Tuberculosis:Pathogenesis, Protection and Control, American Society for Microbiology,Washington, D.C. [1994], pages 85-110). Traditional methods, includingcultivation of the organism require the time and facilities forprolonged incubation. Assays based on DNA amplification are expensiveand technically demanding, and may not be applicable for routine use inclinical laboratories outside of major medical centers in industrializedcountries. The development of a simple, rapid, diagnostic test whichdoes not rely on the growth of organisms in vitro, but that is capableof identifying individuals with latent, subclinical M. tuberculosisinfection, and which might predict the likelihood of subsequent relapse,would be of tremendous value for tuberculosis control programsworldwide.

Monitoring of Therapy

Many factors can adversely affect the response to anti-tuberculoustherapy. These include primary drug resistance, patient non-compliance,malabsorption, adverse interactions with other medications, and otherhost factors. Inadequate treatment can lead to emergence of secondarydrug resistance due to selective pressures on mycobacterial growth. Inorder to assess a patient's response to anti-tuberculosis therapy,patients must be monitored throughout their treatment regimen.

However, sputum cultures and AFB smears return to negative slowly duringtherapy, such that the proportion of samples which become negative aretypically only 40% at 1 month, 80% at 2 months, and 90-95% at 3 months.Chest radiographs also improve slowly, and may not significantly improveuntil 3 months of treatment. It thus is difficult to identify promptlythose patients who ultimately will fail any given tuberculosis treatmentregimen. Better early indicators of success or failure clearly areneeded.

Mitchison has suggested that the early bactericidal activity ofanti-tuberculosis regimens, as determined by quantitative sputumcultures, might predict the overall effectiveness of a treatment regimen(S. L. Chan et al., “The early bactericidal activity of rifabutinmeasured by sputum viable counts in Hong Kong patients with pulmonarytuberculosis,” Tubercle 1992;33-8 [1992]; and A. J. Jindani et al., “TheEarly Bactericidal Activity of Drugs in Patients with PulmonaryTuberculosis,” Am. Rev. Respir. Dis., 121:939-49 [1980]). He observedapproximately a 10⁻³ drop in the number of viable M. tuberculosisbacilli during the first 2 weeks of effective multi-drug therapy, andnoted lesser reductions with less effective regimens. He suggested thatnew drugs for tuberculosis might be evaluated in short term studies (1-2weeks) using quantitative culture as an endpoint. However, this approachhas not been widely accepted, largely because of difficulties ofperforming the assay in a standardized fashion, particularly with regardto homogenization of non-uniform sputum specimens. This lack ofstandardization precludes the use of this method in the clinicalsetting.

Problems also exist in monitoring therapy for MAC infection in AIDSpatients through serial blood cultures. An autopsy series of 44 patientswith MAC bacteremia found that 13 (30%) had no histologic evidence ofMAC disease (F. J. Torriani et al., “Autopsy findings in AIDS patientswith M. avium complex bacteremia,” J. Infect. Dis., 170:1601-5 [1994]),suggesting that transient bloodstream infection may occur and may beself-limited. Conversely, other patients have only transiently orintermittently positive blood cultures in the face of high tissueburdens of mycobacteria (C. A. Kemper et al., “Transient bacteremia dueto M. avium complex in patients with AIDS,” J. Infect. Dis., 170:488-93[1994]). These observations suggest that sustained mycobacteremia may bea late event in the natural history of MAC infection, and that bloodculture is not an adequate diagnostic or monitoring tool when usedalone. However, access to the main site of infection (bone marrow, lymphnode, or liver) requires an invasive diagnostic procedure which is notusually undertaken without prior attempts at diagnosis by non-invasivemeasures. These combined factors often lead to a delay in diagnosis andinitiation of therapy, and make it difficult to evaluate the response totherapy.

In sum, despite advances in the detection of M. tuberculosis and othermycobacteria, the need remains for a safe, reliable, easy-to-use systemfor the detection of infection with these organisms, as well as meansfor monitoring patients with disease or infection. In particular, thereis an urgent need for useful methods to use samples such as urine andother fluids for the detection of infection and disease withMycobacterium species.

SUMMARY OF THE INVENTION

The present invention provides a rapid method for the detection ofdisease and infection due to the mycobacteria, in particular M.tuberculosis, as well as MAC. The present invention is intended fordetection of pulmonary mycobacterial disease or infection, anddisseminated mycobacterial disease, as well as localized infection withmycobacteria at sites other than the pulmonary area.

The present invention provides kits for the detection of Mycobacteriumin a test sample, comprising: a) a solid support; and b) a monoclonalantibody directed against a portion of alpha antigen immobilized on thesolid support. In one embodiment, the kit further comprises a primaryantibody, while in another embodiment the kit comprises a reporterantibody, in yet another embodiment the kit further comprises anamplifier system. It is also contemplated that the kit of the presentinvention comprise a primary antibody and reporter antibody, as well asan amplifier system. In a particularly preferred embodiment, theMycobacterium species detected is Mycobacterium tuberculosis. In analternative embodiment the Mycobacterium species detected isMycobacterium avium.

It is also contemplated that the kit of the present invention willinclude additional components, including, but not limited to, such itemsas an alpha antigen control, a diluent such as saline or water, as wellas a plurality of alpha antigen samples with known concentrations ofalpha antigen suitable for use in preparing a standard curve(s) for thedetermination of antigen concentration in the test (i.e., patient)samples. Furthermore, in kits in which biotinylated antibodies are used,in one preferred embodiment, the primary antibody is preadsorbed with anavidin compound (i.e., strepavidin) coupled to biotinylated captureantibody, prior to use in the kit. It is further contemplated that thekit of the present invention comprise methods for analyzing samplesusing blots, including but not limited to Western blots. These blottingkits may include additional reagents such as those listed above, as wellas reagents, including but not limited to, such as paper suitable forthe blotting system used, and blocking solution.

It is also contemplated that the antibodies of the kit of the presentinvention be prepared through the use of synthetic peptides asimmunogens. In this embodiment, synthetic peptides of known sequence areused to induce the production of at least one of the antibodies used inthe kit. It is also contemplated that the synthetic peptides be used asan immunogen while they are still attached to the beads used to preparethem. Thus, the present invention encompasses antigens of mycobacteriathat are naturally occurring and harvested from samples such as sputum,urine or blood samples, as well as mycobacterial antigens that areproduced synthetically for use in the production of antibodies for usein the kit. It is also contemplated that these synthetic peptides beused as antigens in the kit. In a preferred embodiment, the syntheticpeptides correspond to the alpha antigen of M. tuberculosis or MAC.Thus, the sequences of SEQ ID NOS:1-8 may be used in the form ofsynthetic peptides for use in the present invention. It is alsocontemplated that immune complexes will be tested using the kit of thepresent invention. In this embodiment, the immune complexes may betreated using methods known in the art to dissociate antibodies fromantigens.

It is also contemplated that the samples tested using the kit of thepresent invention will be obtained from individuals infected with one ormore types of the human immunodeficiency virus. It is also contemplatedthat the kit will be used for monitoring the progression of therapy inindividuals infected with Mycobacterium, in particular M. tuberculosisand/or MAC. It is further contemplated that the kit will be used withsamples from patients who are infected with M. tuberculosis, asdetermined by skin test positivity, chest radiograph positivity, and/orsputum cultures containing M. tuberculosis.

The present invention also provides methods for the detection ofMycobacterium in a sample comprising: a) providing: i) a samplesuspected of containing at least a portion of the alpha antigen ofMycobacterium and ii) a monoclonal antibody directed against the portionof Mycobacterium alpha antigen; b) adding the sample to the monoclonalantibody under conditions such that the antibody binds to theMycobacterium alpha antigen in the sample to form an antibody-antigencomplex; and c) detecting the binding of said antigen and antibody.

In a preferred embodiment of the method of the present invention, thedetecting comprises adding the primary antibody to the antigen-antibodycomplex so that the primary antibody binds to the antigen to form anantibody-antigen-antibody sandwich. In another preferred embodiment ofthe method, the detecting further comprises adding a reporter reagent tothe antibody-antigen-antibody sandwich to form anantibody-antigen-antibody-antibody sandwich. In a particularly preferredembodiment, the detecting further comprises adding an amplifier to saidantibody-antigen-antibody-antibody sandwich.

In one embodiment, the monoclonal antibody comprises a murine monoclonalantibody. In a preferred embodiment, the murine monoclonal antibody isbiotinylated. In a particularly preferred embodiment, the primaryantibody is preadsorbed with avidin (e.g., streptavidin) coupled tobiotinylated capture antibody, prior to use in the method of the presentinvention for the detection of Mycobacterium species. In yet anotherparticularly preferred embodiment, the solid support is coated withavidin.

In another embodiment, detection is achieved through use of such methodsas enzyme immunoassay, radioimmunoassay, fluorescence immunoassay,flocculation, particle agglutination, and in situ chromogenic assay. Itis not intended that the present invention be limited to any particularassay format.

It is also contemplated that the method of the present invention willinclude additional components, including, but not limited to, such itemsas an alpha antigen control, a diluent such as saline or water, as wellas a plurality of alpha antigen samples with known concentrations ofalpha antigen suitable for use in preparing standard curve(s) for thedetermination of antigen concentration in the test (i.e., patient)samples. Furthermore, in methods in which biotinylated antibodies areused, in a preferred embodiment, the primary antibody is preadsorbedwith an avidin compound (i.e., strepavidin) coupled to biotinylatedcapture antibody prior to use in the method. It is further contemplatedthat the method of the present invention comprise methods for analyzingsamples using blots, including but not limited to Western blots. Theseblotting methods may include additional reagents such as those listedabove, as well as reagents, including but not limited to, such as papersuitable for the blotting system used, and blocking solution.

It is also contemplated that the antibodies of the method of the presentinvention be prepared through the use of synthetic peptides asimmunogens. In this embodiment, synthetic peptides of known sequence areused to induce the production of at least one of the antibodies used inthe method. It is also contemplated that the synthetic peptides be usedas an immunogen while they are still attached to the beads used toprepare them. Thus, the present invention encompasses antigens ofmycobacteria that are naturally occurring and harvested from samplessuch as sputum, urine or blood samples, as well as mycobacterialantigens that are produced synthetically for use in the production ofantibodies for use in the method. It is also contemplated that thesesynthetic peptides be used as antigens in the kit. In a preferredembodiment, the synthetic peptides correspond to the alpha antigen of M.tuberculosis or MAC. Thus, the sequences of SEQ ID NOS:1-8 may be usedin the form of synthetic peptides for use in the present invention.

In one embodiment of the method, the Mycobacterium detected by themethod of the present invention is selected from the group consisting ofMycobacterium tuberculosis, Mycobacterium avium and Mycobacteriumintracellulare. In one embodiment of the methods of the presentinvention, the portion of said Mycobacterium alpha antigen is selectedfrom the group comprising SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 and 8. Thus,it is contemplated that fragments of the alpha antigen, as well as theentire alpha antigen will be used in the methods of the presentinvention. It is also contemplated that immune complexes will be testedusing the methods of the present invention. In this embodiment, theimmune complexes may be treated using methods known in the art todissociate antibodies from antigens.

It is contemplated that any sample will be used in the method of thepresent invention, including but not limited to urine samples, sputumsamples, blood samples, and serum samples. In the case of sputumsamples, it is contemplated that most samples will be digested and/ordecontaminated using standard methods prior to their analysis in themethod of the present invention. It is also contemplated that somesamples tested using the method of the present invention will beobtained from individuals infected with human immunodeficiency virus,including, but not limited to, HIV-1 and HIV-2.

It is also contemplated that the methods of the present invention willbe used for monitoring the progression of therapy in individualsinfected with Mycobacterium, in particular M. tuberculosis and/or MAC.It is further contemplated that the methods will be used with samplesfrom patients who are infected with M. tuberculosis, as determined byskin test positivity, chest radiograph positivity, and/or sputumcultures containing M. tuberculosis.

The present invention also provides methods for the detection ofmycobacterial antigen in a urine sample comprising a) providing: i) aurine sample suspected of containing at least a portion of alpha antigenof Mycobacterium; and ii) a monoclonal antibody directed against theportion of Mycobacterium alpha antigen; b) adding the urine sample tothe monoclonal antibody under conditions such that the antibody binds tothe Mycobacterium alpha antigen in the urine sample to form anantibody-antigen complex; and c) detecting the binding.

The present invention also provides methods for the detection ofMycobacterium in a sample comprising: a) providing: i) a samplesuspected of containing at least a portion of the alpha antigen ofMycobacterium and ii) a monoclonal antibody directed against the portionof Mycobacterium alpha antigen; b) adding the sample to the monoclonalantibody under conditions such that the antibody binds to theMycobacterium alpha antigen in the sample to form an antibody-antigencomplex; and c) detecting the binding of said antigen and antibody.

In a preferred embodiment of the method of the present invention, thedetecting comprises adding the primary antibody to the antigen-antibodycomplex so that the primary antibody binds to the antigen to form anantibody-antigen-antibody sandwich. In another preferred embodiment ofthe method, the detecting further comprises adding a reporter reagent tothe antibody-antigen-antibody sandwich to form anantibody-antigen-antibody-antibody sandwich. In a particularly preferredembodiment, the detecting further comprises adding an amplifier to saidantibody-antigen-antibody-antibody sandwich.

In one embodiment, the monoclonal antibody comprises a murine monoclonalantibody. In a preferred embodiment, the murine monoclonal antibody isbiotinylated. In a particularly preferred embodiment, the primaryantibody is preadsorbed with avidin (e.g., streptavidin) coupled tobiotinylated capture antibody prior to use in the method of the presentinvention for the detection of Mycobacterium species. In yet anotherparticularly preferred embodiment, the solid support is coated withavidin.

In another embodiment, detection is achieved through use of such methodsas enzyme immunoassay, radioimmunoassay, fluorescence immunoassay,flocculation, particle agglutination, and in situ chromogenic assay. Itis not intended that the present invention be limited to any particularassay format.

It is also contemplated that the method of the present invention willinclude additional components, including, but not limited to, such itemsas an alpha antigen control, a diluent such as saline or water, as wellas a plurality of alpha antigen samples with known concentrations ofalpha antigen suitable for use in preparing standard curve(s) for thedetermination of antigen concentration in the test (i.e., patient)samples. Furthermore, in methods in which biotinylated antibodies areused, in a preferred embodiment, the primary antibody is preadsorbedwith an avidin compound (i.e., strepavidin) coupled to biotinylatedcapture antibody prior to use in the method. It is further contemplatedthat the method of the present invention comprise methods for analyzingsamples using blots, including but not limited to Western blots. Theseblotting methods may include additional reagents such as those listedabove, as well as reagents, including but not limited to, such as papersuitable for the blotting system used, and blocking solution.

It is also contemplated that the antibodies of the method of the presentinvention be prepared through the use of synthetic peptides asimmunogens. In this embodiment, synthetic peptides of known sequence areused to induce the production of at least one of the antibodies used inthe method. It is also contemplated that the synthetic peptides be usedas an immunogen while they are still attached to the beads used toprepare them. Thus, the present invention encompasses antigens ofmycobacteria that are naturally occurring and harvested from samplessuch as sputum, urine or blood samples, as well as mycobacterialantigens that are produced synthetically for use in the production ofantibodies for use in the method. It is also contemplated that thesesynthetic peptides be used as antigens in the kit. In a preferredembodiment, the synthetic peptides correspond to the alpha antigen of M.tuberculosis or MAC. Thus, the sequences of SEQ ID NOS:1-8 may be usedin the form of synthetic peptides for use in the present invention.

In one embodiment of the method, the Mycobacterium detected by themethod of the present invention is selected from the group consisting ofMycobacterium tuberculosis, Mycobacterium avium and Mycobacteriumintracellulare. In one embodiment of the methods of the presentinvention, the portion of said Mycobacterium alpha antigen is selectedfrom the group comprising SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 and 8. Thus,it is contemplated that fragments of the alpha antigen, as well as theentire alpha antigen will be used in the methods of the presentinvention. It is also contemplated that immune complexes present inurine samples will be tested using the methods of the present invention.In this embodiment, the immune complexes may be treated using methodsknown in the art to dissociate antibodies from antigens.

It is also contemplated that some samples tested using the method of thepresent invention will be obtained from individuals infected with humanimmunodeficiency virus, including, but not limited to, HIV-1 and HIV-2.

It is also contemplated that the methods of the present invention willbe used for monitoring the progression of therapy in individualsinfected with Mycobacterium, in particular M. tuberculosis and/or MAC.It is further contemplated that the methods will be used with samplesfrom patients who are infected with M. tuberculosis, as determined byskin test positivity, chest radiograph positivity, and/or sputumcultures containing M. tuberculosis.

The present invention also provides a composition comprising purifiedmonoclonal antibody directed against a portion of Mycobacterium alphaantigen. In one embodiment, the monoclonal antibody is a murinemonoclonal antibody. In an alternative embodiment, the murine monoclonalantibody is directed against epitopes of the alpha antigen of M.tuberculosis, M. avium or M. intracellulare. In a particularly preferredembodiment, the murine monoclonal antibody is directed against M.tuberculosis epitopes. In yet another preferred embodiment, the alphaantigen used to produce the murine monoclonal antibody is comprised ofSEQ ID NOS:1, 2, 3, 4, 5, 6, 7, or 8. It is also contemplated that themonoclonal antibody of the present invention will be produced throughthe use of synthetic peptides as antigens. In a preferred embodiment,the monoclonal antibody of the present invention is raised against SEQID NOS: 3, 4, 6 or 7. It is further contemplated that the monoclonalantibody be produced using any suitable methods for monoclonal antibodyproduction, including intrasplenic methods, cell culture, etc. It is notintended that the monoclonal antibody of the present invention belimited to an antibody produced using a particular method or producedusing a particular animal species. It is also not intended that theantibodies used in the present invention be of a particular class ofimmunoglobulin.

DESCRIPTION OF THE FIGURES

FIG. 1 shows Western blots for detection of alpha antigen purified fromculture filtrates.

FIG. 2 is a graphical representation of the reaction used to produce acolored reaction product in the amplified ELISA test method.

FIG. 3 is a graph showing the results of an amplified ELISA test fordetection of alpha antigen as described in Example 3.

FIG. 4 is a graph showing the detection of alpha antigen in spikedsamples of human serum and urine.

FIG. 5 is a graph showing the results of a comparison between detectionof M. tuberculosis by the BACTEC system and the amplified ELISA testmethod.

FIG. 6 is a graph showing the amplified ELISA results for urine samplesfrom HIV-negative tuberculosis patients and controls.

FIG. 7 is a graph showing the amplified ELISA results for serum samplesfrom HIV-negative tuberculosis patients and controls.

FIG. 8 is a graph showing the amplified ELISA results for urine samplesfrom HIV-positive tuberculosis patients and controls.

FIG. 9 shows Western blots of filtrates of M. tuberculosis, M. avium,and M. smegmatis using monoclonal TBC27 as the capture antibody.

FIG. 10 is a graph showing the standard curves for alpha antigenamplified ELISA using culture filtrates of M. tuberculosis and M. avium.

FIG. 11 shows the urinary antigen values in HIV-negative subjectsgrouped according to their M. tuberculosis and M. avium status.

FIG. 12 shows the urinary antigen values in HIV-positive subjectsgrouped according to their M. tuberculosis and M. avium status.

FIGS. 13A-13C show the results of alpha antigen detection assays forsputum samples obtained from three patients, with one patient's resultsbeing shown in each of the three (i.e., FIGS. 13A-13C).

FIGS. 14A-14F show the results of alpha antigen detection assays forsputum samples obtained from nine patients, with one patient's resultsbeing shown in each of the nine figures (i.e., FIGS. 14A-14F).

FIG. 15 shows the results of alpha antigen detection assays for sputumsamples obtained from one patient.

FIG. 16 shows Western blots for the detection of alpha antigen by threemonoclonal antibodies.

FIG. 17 shows the results of alpha antigen detection assays from urinesamples of HIV-positive and HIV-negative individuals.

FIG. 18 shows a Western blot in which 5C5 was used to detect alphaantigen.

FIG. 19 shows the amino acid sequences of M. tuberculosis and M. aviumalpha antigen.

FIG. 20 shows Western blots of M. avium filtrate tested with murineantiserum directed against alpha antigen peptides.

FIGS. 21A-21C show Western blots of M. avium and M. tuberculosis culturefiltrates tested with goat M. intracellulare antisera. FIG. 21A showsthe results with Kris antiserum; FIG. 21B shows the results with Krisantiserum adsorbed with M. tuberculosis; and FIG. 21C shows the resultswith Jane antiserum.

FIGS. 22A-22B show Western blots of M. avium and M. tuberculosis culturefiltrates tested with Kris antiserum and Kris antiserum adsorbed with M.tuberculosis. FIG. 22A shows the results with the unabsorbed antiserumand FIG. 22B shows the results with adsorbed antiserum.

GENERAL DESCRIPTION OF THE INVENTION

Previous reports of antigen detection assays for direct diagnosis oftuberculosis have been limited to examination of fluids obtained fromthe site of clinical disease, such as cerebrospinal fluid, sputum, orbronchoalveolar lavage fluid. Furthermore, the range of the lower limitof detection of these assays was reported to be from 1 ng to 1 μg/ml. Inaddition, the lower detection thresholds were only achieved in thoseassay which used a polyclonal antiserum for capture. Moreover, severalused the same serum for both capture and detection. In none of thesereports were fluids remote from the site of infection studied. None ofthese assays were able to identify subjects with latent infection. Thisis in contrast with the present invention, in which identification of amycobacterial antigen in fluids remote from the site of infection inpatients infected with M. tuberculosis is possible.

In the method of the present invention, a sandwich ELISA (enzyme-linkedimmnuosorbent assay) with a monoclonal antibody for capture and asecondary antibody for detection of bound monoclonal antibody is used.In a preferred embodiment, the signal from the assay is amplified usingan recycling enzymatic method, which increases the signal over 500 fold.Special measures are included in order to reduce background noise. Theassay offers numerous advantages as compared to existing methods fortuberculosis diagnosis. First, body fluids remote from the site ofinfection can be tested using the method of the present invention. Thetest can be used with infected body fluids, as well as urine and blood.Although urine and blood usually do not contain M. tuberculosis, thesesites may contain secreted products of the infection. Detection of thesesecreted products provides a clear advantage in attempts to diagnose alocalized infection such as is usually the case in tuberculosis. It is aparticularly important advantage in diagnosis of tuberculosis inchildren, from whom quality sputum specimens are difficult to collectand are generally not available.

Detection of Alpha Antigen

Alpha antigen is an abundantly-produced, secreted 30 kD mycobacterialprotein involved in mycolic acid synthesis. During development of thepresent invention, it was determined that alpha antigen was apotentially useful indicator of mycobacterial disease, as it can accountfor up to 20% of the protein content of spent mycobacterial culturemedium (A. Andersen et al., “Proteins released from Mycobacteriumtuberculosis during growth,” Infect. Immun., 59:1905-10 [1991]). Inaddition, homologous alpha proteins exist for most species ofmycobacteria, including the M. tuberculosis and M. avium complexes.However, it is also contemplated that other antigens may be detectedusing the methods and compositions of the present invention.

The present invention provides rapid, reliable detection ofmycobacterial antigens in such samples as body fluids (e.g., urine,blood and sputum) for the detection of various mycobacterial species, asthe antigens have both species specific and shared epitopes (H. Tasakaet aL, “Specificity and distribution of alpha antigens of Mycobacteriumavium-intracellulare, Mycobacterium scrofulaceum, and related species ofmycobacteria,” Am. Rev. Respir. Dis., 1985;132:173-4 [1985]).Furthermore, the genes for alpha antigens of M. tuberculosis, bovis,avium, and kansasii, have been cloned and sequenced (See e.g., K. Matsuoet al., “Cloning and expression of the gene for the cross-reactive alphaantigen of Mycobacterium kansasii,” Infect. Immun., 58:550-6 [1990]; K.Matsuo et al., “Cloning and expression of the Mycobacterium bovis BCGgene for extracellular alpha antigen,” J Bacteriol., 170:3847-54 [1988];N. Ohara et al., “Cloning and sequencing of the gene for alpha antigenfrom M. avium and mapping of B-cell epitopes,” Infect. Immun., 61:1173-9[1993]; and L. DeWit et al., “Nucleotide sequence of the 85B-proteingene of M. bovis BCG and M. tuberculosis: DNA sequence,” J. DNA Seq.Map., in press. [1996]).

At 30 kD), the alpha antigens may be too large to be filtered intactthrough the renal glomeruli, particularly when complexed with antibody,nonetheless, the present invention provides means to detect alphaantigens present in urine samples. While an understanding of themechanism by which it is possible to detect the antigens in urine is notnecessary to the successful practice of the invention, it is believedthat cleavage of the protein may occur in vivo as a consequence ofintracellular digestion by proteolytic enzymes within the macrophages,in the circulation, or in the kidney (e.g., by brush border peptidases),and permit excretion of the antigen or portions thereof. Antibodyepitopes of alpha antigens of M. bovis, M. leprae, M. tuberculosis, andM. avium are largely restricted to the carboxy-terminal half of theprotein (Ohara et al., “Cloning and sequencing of the gene for alphaantigen from M. avium and mapping of B-cell epitopes,” Infect. Immun.,61:1173-9 [1993]; and E. Filley et al., “Identification of an antigenicdomain on Mycobacterium leprae protein antigen 85B, which isspecifically recognized by antibodies from patients with leprosy,” JInfect. Dis., 169:162-9 [1994]). Thus, fragments representing theN-terminal portion of M. tuberculosis may be present in the urine, evenif the native antigen is too large to be filtered by the glomeruli. Itis contemplated that these fragments will be detected using thecompositions and methods of the present invention.

The present invention provides numerous advantages over the methodspreviously and currently in use to detect infection and/or disease withM. tuberculosis or MAC. For example, all of the previously reportedtuberculosis antigen detection assays were applied only to infectedfluids or to early cultures, the assays were considerably less sensitivethan this assay, and in addition, relied upon an animal antiserumdeveloped against a whole organism of another mycobacterial species(BCG) for detection, resulting in poor specificity. Furthermore, none ofthe previously described assays were applied to urine and blood samples.

Also, the present invention detects products of the mycobacteria, ratherthan the host response to the organism. This provides an importantadvantage, as the host response is impaired in immunocompromisedpatients at highest risk of tuberculosis. Thus, tests such as thosepreviously reported that are based on the host response areinsufficiently sensitive in this population. In addition, the presentinvention permits detection of latent infection in individuals athighest risk of developing tuberculosis. This is significant, becauseonce the infection is detected, preventive therapy can be initiated.This is not possible with the previously described methods.

Also, unlike the previously described methods, the present invention canbe used for early monitoring of anti-tuberculosis therapy, providing auseful tool in predicting which individuals might experience treatmentfailure with a particular regimen. Thus, the present invention can alsobe used in the rapid evaluation of new anti-tuberculosis therapies.

Importantly for clinical diagnosis and public health tuberculosisclinics, the method of the present invention is rapid, requiring only1-2 days to complete, as opposed to a minimum of 2-4 weeks for culture.Furthermore, the present invention can be performed during a singlevisit to the clinic, unlike the routine skin testing methods whichrequire a return visit in order to observe and record the skin testresults. Also importantly, the present invention provides an inexpensivemethod that is within the capabilities of existing clinical and publichealth mycobacteria laboratories. This is generally not true for methodsthat require specialized equipment and training (e.g., the polymerasechain reaction [PCR]). The test is very flexible, permitting thedetection of various antigens or alpha antigen epitopes. The assay canbe used to detect all secreted mycobacterial antigens by substituting anon-specific binding method for the capture antibody.

In addition, the results obtained by the present invention for detectionof antigenuria are much superior to those of such previous methods asthat of Sippola et al. (A. A. Sippola et al., “Mycobacterium aviumantigenuria in patients with AIDS and disseminated M. avium disease,” J.Infect. Dis., 168:466-8 [1993]). For example, as described in Example 16of the present application, the K-II antiserum was found by Western blotof M. avium filtrate, to primarily recognize three antigens. One antigenwas approximately 38-42 kD, and may be lipoarabinomannan (LAM), amycobacterial polysaccharide. The second antigen is a 30 kD antigen thatwas determined to be alpha antigen. The third antigen was approximately25 kD. This is significant for the reliable and reproducible detectionof antigenuria, as these antigens differ considerably in terms ofpotential species-specificity. Antibodies to polysaccharides reach highlevels in mycobacterial infection; however, they generally are broadlycross reactive and have limited diagnostic potential (S. D. Chaparas etal., “Tuberculin-active carbohydrate that induces inhibition ofmacrophage migration but not lymphocyte transformation,” Science170:637-9 [1970]; and A. Drowart et al., “Isoelectrophoreticcharacterization of protein antigens present in mycobacterial culturefiltrates and recognized by monoclonal antibodies directed against theMycobacterium bovis BCG antigen 85 complex,” Scand. J. Immunol.,36:697-702 [1992]). It is contemplated that in addition to the alphaantigen, antigens such as the 25 kD antigen described in Example 16 willbe detected in the present invention.

In contrast with the previous studies, the present invention provides amore sensitive method for the reliable detection of infection and/ordisease due to M. tuberculosis or MAC, based on the detection ofmycobacterial products, rather than the host response to themycobacteria.

Definitions

To facilitate further understanding of the invention, a number of termsare defined below:

The terms “sample” and “specimen” in the present specification andclaims are used in their broadest sense. On one hand, they are meant toinclude a specimen or culture. On the other hand, they are meant toinclude both biological and environmental samples. These termsencompasses all types of samples obtained from humans and other animals,including but not limited to, body fluids such as urine, blood, sputum,fecal matter, cerebrospinal fluid (CSF), semen, and saliva, as well assolid tissue. These terms also refers to swabs and other samplingdevices which are commonly used to obtain samples for culture ofmicroorganisms.

Biological samples may be animal, including human, fluid or tissue, foodproducts and ingredients such as dairy items, vegetables, meat and meatby-products, and waste. Environmental samples include environmentalmaterial such as surface matter, soil, water, and industrial samples, aswell as samples obtained from food and dairy processing instruments,apparatus, equipment, disposable, and non-disposable items. Theseexamples are not to be construed as limiting the sample types applicableto the present invention.

Whether biological or environmental, a sample suspected of containingmicroorganisms may (or may not) first be subjected to an enrichmentmeans to create a “pure culture” of microorganisms. By “enrichmentmeans” or “enrichment treatment,” the present invention contemplates (i)conventional techniques for isolating a particular microorganism ofinterest away from other microorganisms by means of liquid, solid,semi-solid or any other culture medium and/or technique, and (ii) noveltechniques for isolating particular microorganisms away from othermicroorganisms. It is not intended that the present invention be limitedonly to one enrichment step or type of enrichment means. For example, itis within the scope of the present invention, following subjecting asample to a conventional enrichment means, to subject the resultantpreparation to further purification such that a pure culture of a strainof a species of interest is produced. This pure culture may then beanalyzed by the medium and method of the present invention.

As used herein, the term “culture” refers to any sample or specimenwhich is suspected of containing one or more microorganisms. “Purecultures” are cultures in which the organisms present are only of onestrain of a particular genus and species. This is in contrast to “mixedcultures,” which are cultures in which more than one genus and/orspecies of microorganism are present.

As used herein, the term “organism” is used to refer to any species ortype of microorganism, including but not limited to bacteria, yeasts andother fungi. As used herein, the term fungi, is used in reference toeukaryotic organisms such as the molds and yeasts, including dimorphicfingi.

As used herein, the term “Mycobacterium” is used in reference to thegenus Mycobacterium, the only genus in the family Mycobacteriaceae. Asused herein, the term “mycobacteria” is used in reference to all of theorganisms included within the genus Mycobacterium, including all of thetaxonomic levels lower than genus, including, but not limited tospecies, subspecies, strains, etc. As used herein, M. tuberculosis isused in reference to all of the species included within the “M.tuberculosis complex,” including M. tuberculosis, M. bovis, M. microti,M. africanum, and any other organism subsequently recognized as fallingwithin this taxonomic group.

As used herein, the terms “microbiological media” and “culture media,”and “media” refer to any substrate for the growth and reproduction ofmicroorganisms. “Media” may be used in reference to solid plated mediawhich support the growth of microorganisms. Also included within thisdefinition are semi-solid and liquid microbial growth systems includingthose that incorporate living host organisms, as well as any type ofmedia.

As used herein, the term “primary isolation” refers to the process ofculturing organisms directly from a sample. Thus, primary isolationinvolves such processes as inoculating an agar plate from a cultureswab, urine sample, environmental sample, etc. Primary isolation may beaccomplished using solid or semi-solid agar media, or in liquid. As usedherein, the term “isolation” refers to any cultivation of organisms,whether it be primary isolation or any subsequent cultivation, including“passage” or “transfer” of stock cultures of organisms for maintenanceand/or use.

As used herein, the term “presumptive diagnosis” refers to a preliminarydiagnosis which gives some guidance to the treating physician as to theetiologic organism involved in the patient's disease. Presumptivediagnoses are often based on “presumptive identifications,” which asused herein refer to the preliminary identification of a microorganismbased on observation such as colony characteristics, growth on primaryisolation media, gram stain results, etc.

As used herein, the term “definitive diagnosis” is used to refer to afinal diagnosis in which the etiologic agent of the patient's diseasehas been identified. The term “definitive identification” is used inreference to the fmal identification of an organism to the genus and/orspecies level.

As used herein, the terms “digestion” and “decontamination” are used inreference to the standard methods used by mycobacteriology laboratoriesto treat samples such as sputum prior to culturing or testing thesamples.

As used herein, the term “antibody” is used in reference to anyimmunoglobulin molecule that reacts with a specific antigen. It isintended that the term encompass any immunoglobulin (e.g., IgG, IgM,IgA, IgE, IgD, etc.) obtained from any source (e.g., humans, rodents,non-human primates, caprines, bovines, equines, ovines, etc.).

As used herein, the term “antigen” is used in reference to any substancethat is capable of reacting with an antibody. It is intended that thisterm encompass any antigen and “immunogen” (i.e., a substance whichinduces the formation of antibodies). Thus, in an immunogenic reaction,antibodies are produced in response to the presence of an antigen orportion of an antigen.

As used herein, the terms “antigen fragment” and “portion of an antigen”are used in reference to a portion of an antigen. Antigen fragments orportions may occur in various sizes, ranging from a small percentage ofthe entire antigen to a large percentage, but not 100% of the antigen.However, in situations where at least a portion of an antigen isspecified, it is contemplated that the entire antigen may be present. Itis contemplated that antigen fragments or portions, may, but are notrequired to comprise an “epitope” recognized by an antibody. Antigenfragments or portions also may or may not be immunogenic.

As used herein, the term “immunoassay” is used in reference to anymethod in which antibodies are used in the detection of an antigen. Itis contemplated that a range of immunoassay formats be encompassed bythis definition, including but not limited to direct immunoassays,indirect immunoassays, and “sandwich” immunoassays.” A particularlypreferred format is a sandwich enzyme-linked immunosorbent assay(ELISA). However, it is not intended that the present invention belimited to this format. It is contemplated that other formats, includingradioimmunoassays (RIA), immunofluorescent assays (IFA), and other assayformats, including, but not limited to, variations on the ELISA methodwill be useful in the method of the present invention. Thus, otherantigen-antibody reaction formats may be used in the present invention,including but not limited to “flocculation” (ie., a colloidal suspensionproduced upon the formation of antigen-antibody complexes),“agglutination” (i.e., clumping of cells or other substances uponexposure to antibody), “particle agglutination” (i.e., clumping ofparticles coated with antigen in the presence of antibody or theclumping of particles coated with antibody in the presence of antigen);“complement fixation” (ie., the use of complement in an antibody-antigenreaction method), and other methods commonly used in serology,immunology, immunocytochemistry, histochemistry, and related fields.

As used herein, the term “cell staining” is used in reference to methodsused to label or stain cells to enhance their visualization. Thisstaining or labelling may be achieved through the use of variouscompounds, including but not limited to, fluorochromes, enzymes, gold,and iodine. It is contemplated that the definition encompasses suchmethods as “in situ chromogenic assays,” in which a test (i.e., anassay) is conducted on a sample in situ. It is also contemplated thatthe in situ chromogenic assay will involve the use of an immunoassay(i.e., an ELISA).

As used herein, the term “capture antibody” refers to an antibody thatis used to bind an antigen and thereby permit the recognition of theantigen by a subsequently applied antibody. For example, the captureantibody may be bound to a microtiter well and serve to bindmycobacterial antigens present in a sample added to the well. Anotherantibody (termed the “primary antibody”) is then used to bind to theantigen-antibody complex, in effect to form a “sandwich” comprised ofantibody-antigen-antibody. Detection of this complex can be performed byseveral methods. The primary antibody may be prepared with a label suchas biotin, an enzyme, a fluorescent marker, or radioactivity, and may bedetected directly using this label. Alternatively, a labelled “secondaryantibody” or “reporter antibody” which recognizes the primary antibodymay be added, forming a complex comprised ofantibody-antigen-antibody-antibody. Again, appropriate reporter reagentsare then added to detect the labelled antibody. Any number of additionalantibodies may be added as desired. These antibodies may also belabelled with a marker, including, but not limited to an enzyme,fluorescent marker, or radioactivity.

As used herein, the term “reporter reagent” or “reporter molecule” isused in reference to compounds which are capable of detecting thepresence of antibody bound to antigen. For example, a reporter reagentmay be a calorimetric substance which is attached to an enzymaticsubstrate. Upon binding of antibody and antigen, the enzyme acts on itssubstrate and causes the production of a color. Other reporter reagentsinclude, but are not limited to fluorogenic and radioactive compounds ormolecules. This definition also encompasses the use of biotin andavidin-based compounds (e.g., including compounds but not limited toneutravidin and streptavidin) as part of the detection system. In oneembodiment of the present invention, biotinylated antibodies may be usedin the present invention in conjunction with avidin-coated solidsupport.

As used herein the term “signal” is used in reference to an indicatorthat a reaction has occurred, for example, binding of antibody toantigen. It is contemplated that signals in the form of radioactivity,fluorogenic reactions, and enzymatic reactions will be used with thepresent invention. The signal may be assessed quantitatively as well asqualitatively.

As used herein, the term “amplifier” is used in reference to a systemwhich enhances the signal in a test method such as an ELISA.

As used herein, the term “solid support” is used in reference to anysolid material to which reagents such as antibodies, antigens, and othercompounds may be attached. For example, in the ELISA method, the wellsof microtiter plates often provide solid supports. Other examples ofsolid supports include microscope slides, coverslips, beads, particles,cell culture flasks, as well as many other items.

As used herein, the term “kit” is used in reference to a combination ofreagents and other materials. It is contemplated that the kit mayinclude reagents such as capture antibody, reporter antibody, andamplifier.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for the rapiddetection of mycobacterial antigens in such samples as blood, serum,urine, sputum, and culture filtrates. Therefore, this invention providessignificant advantages in the diagnosis, monitoring, and treatment ofpatients with infections and/or disease with such organisms as M.tuberculosis and MAC.

In particular, the present invention provides methods and compositionsfor an amplified ELISA method for the detection of the mycobacteria. Inthis method, two antibody preparations are used, namely a “capture”antibody and a “reporter” antibody (or “antibody pair”). The captureantibody is used to “capture” mycobacterial antigens present in asample, in order to permit their detection upon the addition of thereporter antibody and amplifier system.

In a particularly preferred embodiment, the present invention providescompositions and methods for the detection of alpha antigen present inpatient samples or other specimens. It is contemplated that the methodbe used to detect either the antigen or specific epitopes within thealpha antigen. It is not intended that the invention be limited toparticular epitopes of the alpha antigen. Thus, it is also not intendedthat the present invention be limited to particular antibodies. Forexample, the capture antibody may be a monoclonal antibody directedagainst alpha antigen of M. tuberculosis, while the reporter antibody isa polyclonal or monoclonal antibody directed against an organism, suchas M. bovis BCG.

Although embodiments have been described with some particularity, manymodifications and variations of the preferred embodiment are possiblewithout deviating from the invention.

Experimental

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); pg (picograms); l or L (liters); ml (milliliters); μl(microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm(nanometers); OD (optical density); OD₄₉₂ (optical density at 492 nm);°C. (degrees Centigrade); PPD (purified protein derivative of M.tuberculosis, Connaught Laboratories, Ontario, Canada); PBS (phosphatebuffered saline, Sigma); CHAPS detergent(3[3(3-Cholamidopropyll)diethylammonio]-1-propane-sulfonate); BSA(bovine serum albumin, Pierce); Tris (TRIZMA, Sigma); RPM (revolutionsper minute); TBS (Tris buffered saline 0.05 M pH 8.0, Sigma). Thefollowing commercial enterprises or governmental agencies are identifiedas follows: Amersham (Amersham Life Science, Arlington Hts, Ill.); ATCC(American Type Culture Collection, Rockville, Md.); Becton Dickinson(Becton Dickinson Diagnostic Systems Instrument Systems, Sparks, Md.;Becton Dickinson Microbiology Systems, Cockeysville, Md.); BioRad(BioRad, Hercules, Calif.); Biowhittaker (Walkersville, Md.); BoehringerMannheim (Indianapolis, Ind.); DAKO (DAKO, Carpinteria, Calif.); Difco(Difco Laboratories, Detroit, Mich.); Fisher (Fisher Scientific,Pittsburgh, Pa.); Isolator (Wampole Laboratories, Cranbury, N.J.); NCBI(National Center for Biotechnology Information, Bethesda Md.,http://www3.ncbi.nlm.nih.gov/Entrez/index.html); NLM (National Libraryof Medicine, Bethesda Md.); Pierce (Pierce, Rockford, Ill.); S&S(Schleicher & Schuell, Keene, N.H.); Sigma (Sigma Chemical Co., St.Louis, Mo.); PGC (PGC Scientifics, Gaithersburg, Md.); Spectrum(Spectrum Medical Industries, Los Angeles, Calif.); U.S. Biochemical(U.S. Biochemical Corp., Cleveland, Ohio); Cambridge (CambridgeDiagnostics, Cambridge, Mass.); and Molecular Devices (MolecularDevices, Menlo Park, Calif.). Unless otherwise indicated, all of thechemicals used in the following Examples were obtained from Sigma.

EXAMPLE 1 Growth of Mycobacterium tuberculosis, Preparation of CultureFiltrates, Purification of Alpha Antigen, and Confirmation by WesternBlot

In this Example, cultures of M. tuberculosis were grown in order toproduce culture filtrates for detection of alpha antigen. M.tuberculosis strain H37Rv was obtained from ATCC. Although ATCC #27294was utilized in this Example, it is contemplated that other deposits ofH37Rv (e.g., ATCC #25618, 35800, etc.) will be suitable for use in thepresent invention. Bacilli were cultured in Proskauer Beck medium inroller bottles at 37° C., at 5 RPM, in a humidified atmosphere for 10weeks. Bacilli were removed by filtration using filter paper withprogressively smaller pore size filters, up to 0.2μ (S & S). Ammoniumsulfate (Sigma) was added to the liquid spent medium to a fmalconcentration of 60% saturation at 3° C. The filtrate was refrigeratedwith constant magnetic stirring overnight. The resulting precipitate wassedimented at 2000 RPM. The liquid was removed, the precipitateresuspended in distilled water, and then was dialysed against a waterbath using a 10 kD cutoff dialysis membrane (Spectrum).

The precipitated protein was applied to a diethylaminoethyl cellulosecolumn (Sigma), washed with 0.01 M phosphate buffer, and then elutedwith a linear gradient of from 0.01 M to 0.3 M sodium phosphate. Alphaantigen eluted at a salt concentration of 0.07 M. The antigen wasconcentrated by lyophilization, and then applied to a preparativeisoelectric focusing cell (Rotofor chamber, Biorad), with 10 mg CHAPSdetergent (Bio-Rad), 5 ml glycerol, and 1 ml pH 3-10 ampholytes(Bio-Rad), 1 ml pH 5-7 ampholytes (Bio-Rad) and a final volume of 55 ml.A constant power of 12 W was applied for 5 hr. The fractions containingalpha antigen were identified by SDS-PAGE and Western blot as describedbelow. Selected fractions were then further fractionated by preparativeSDS-PAGE using a PrepCell column (Bio-Rad) using a 10% acrylamide gelaccording to the manufacturer's recommendations. Protein fractions werecollected from this column and were simultaneously concentrated anddialyzed using a cylindrical membrane device with a 10 kD cutoff(Micro-ProDiCon, Spectrum). Fractions containing pure alpha antigen wereidentified by SDS-PAGE, colloidal gold stain (Aurodye, Amersham), andWestern blot.

SDS polyacrylamide gel electrophoresis was performed using 10%acrylarnide in the running gel, and 4% acrylamide in the stacking gel,in a Mini-Protean apparatus (Biorad) as suggested by the manufacturer.The protein to be electrophoresed was mixed with an equal volume ofreducing sample buffer (Tris 1.51 g, pH 6.75, 4% SDS, 20% glycerol, and10 μl 2-mercaptoethanol suspended to 100 ml in H₂O), and boiled for 2minutes prior to application to the gel. After electrophoresis, thesample was transferred to 0.2μ pore size nitrocellulose paper (S&S).

Western blotting was performed by blocking nonspecific binding with 5%bovine fetal serum (BioWhittaker) and 5% bovine serum albumin (BSA,Sigma) in phosphate buffered saline (Sigma). The antibody was dilutedfrom 1:10 to 1:5000, as needed (i.e., depending upon the startingconcentration), in wash buffer with 1% BSA, and was incubated with thenitrocellulose blot overnight with constant rocking at 0.5 RPM at roomtemperature. The blot was washed twice for 5 minutes in PBS with Tween20 0.1% (Sigma). An alkaline phosphatase conjugated secondary antibody(Sigma) was diluted 1:500 in PBS with 1% BSA and incubated for 6 hr withrocking at room temperature. The blot was washed twice as above. Theblot was then added to 30 ml of a solution of 100 mM Tris, 100 mM NaCl,5 mM MgCl₂, with 50 mg nitroblue tetrazolium and 5 mg5-bromo-4-chloro-3-indoyl phosphate. Development was stopped by washingin water.

These methods were satisfactory for purification of alpha antigen (85-B)from its related antigen 85-A, which differs only slightly in molecularsize but more substantially by isoelectric point. A blot of M.tuberculosis alpha antigen purified by this approach is shown in FIG. 1.In this Figure, lane “a” contained M. tuberculosis culture filtrate asthe antigen and the stain on this lane was colloidal gold. Lane “b” ofFIG. 1 contained alpha antigen purified as described above and the stainwas also colloidal gold. Lane “c” of FIG. 1 contained alpha antigenidentical to lane b and stained with biotinylated TBC27.

EXAMPLE 2 Production of Murine Monoclonal Antibody

The monoclonal antibody designated as TBC27 was produced for use in theELISA method of the present invention. This monoclonal was developed byimmunization with M. tuberculosis alpha antigen obtained using themethods of Example 1. Six week-old female Balb/c mice (Charles River,Mass.) were immunized with 50 μg alpha antigen in incomplete Freund'sadjuvant (DIFCO, Detroit, Mich.). Twice, animals were boosted withadditional 50 μg of antigen at two week intervals, and sacrificed 2weeks after the last boost. Spleen cells were fused to SP2/0 Ag 14myeloma cells (ATCC) using polyethylene glycol (Sigma), and werepropagated in Dulbecco's modified Eagle' medium (Sigma) with addedhypoxanthine, aminopterin, and thymidine. Supernatants of wells withvisible growth were tested for antibody by ELISA. The wells of Immulon 2plates (PGC) were sensitized overnight (i.e., 12-18 hours), with 50 μlM. tuberculosis filtrate (10 μg/ml), and 25 μl of 3% glutaraldehyde in0.15 M sodium phosphate. The plates were washed with wash buffer (TBSwith 0.1% Tween-20). Supernatants from hybridoma wells were added to thewells and allowed to incubate overnight at room temperature, withrocking at 5 RPM. The plates were washed with wash buffer. Antibody wasdetected using alkaline phosphatase-conjugated anti-mouse Ig (Sigma) andalkaline phosphatase, as recommended by the manufacturer (Sigma). Wellsfound to be positive for antibody production were expanded in culture,through the use of standard methods for expansion of monoclonalantibody-producing clones. The supernatants were collected, lyophilized,and dialyzed. The antibody was biotinylated using the ImmunoPure NHSLC-biotin kit (Pierce) and the manufacturer's recommendations.

An immunoadsorbent column was prepared using ImmunoPure immobilizedstrepavidin (Pierce) according to the manufacturer's recommendations.Biotinylated TBC-27 was passed twice over the column. The column wasregenerated between passes, according to the manufacturer'srecommendations. The preadsorbed TBC-27 was used in subsequent studiesfor the antigen detection ELISA, as described below.

EXAMPLE 3 Detection of Alpha Antigen in M. tuberculosis CultureFiltrates

In this Example, an ELISA method (C. J. Stanley et al, “Enzymeamplification can enhance both the speed and the sensitivity ofimmunoassays,” J. Immunol. Meth., 83:89-95 [1985]; and C. H. Self,“Enzyme amplification—a general method applied to provide animmunoassisted assay for placental alkaline phosphatase,” J. Immunol.Meth., 76:389-93 [1985]) that substantially increases the speed andsensitivity of ELISA, was extensively modified for use for the detectionof alpha antigen, including the addition of another step. In thismethod, an alkaline phosphatase conjugate was used to convert NADP toNAD, which then entered a regenerating cycle leading to production of acolored formazan dye. A schematic of the reaction is shown in FIG. 2.Its use in the alpha antigen assay has resulted in over a 1000 foldreduction in the lower limit of detection of the assay (ie., from 20ng/ml, to from 1 to 10 pg/ml). The samples used in this pilot study wereculture filtrates obtained from cultures of M. tuberculosis grown asdescribed in Example 1.

In this method, 100 μl biotinylated TBC27 diluted 1:1000 in BSA blocker(Pierce) in TBS was added to each well of neutravidin-precoated ELISAplates (commercially available from Pierce). After 2 hr, the plate waswashed with wash buffer (TBS/Tween 20 0.1%). Further nonspecific bindingwas inhibited by adding 300 μl SuperBlock (Pierce) twice, as per themanufacturer's recommendations.

Standards (M. tuberculosis filtrate, with a 10% alpha antigen content asdetermined by densitometry of colloidal gold stained gels) and sampleswere diluted with equal volumes of an appropriate buffer depending onthe nature of the experiment. For example, in assays comparingmycobacterial filtrates of different species, or of sputum samples, thediluent was TBS. For serum or urine, the diluent was pooled serum orurine (respectively) obtained from a group of healthy individuals withno known TB exposure, no residence in a TB endemic area, no history ofBCG vaccination, a PPD skin test reaction of 0 mm induration within thepast year, and no history of a reaction of greater than 0 mm in thepast. The diluted standards, and the samples, were then diluted with anequal volume of BSA blocker in TBS. One hundred μl was then added toeach well. Standards were tested in duplicate, and samples were testedin individual wells. The plate was incubated overnight on a rockingtable at 5 RPM in a humidor. The plate was washed 3 times with washingbuffer. 100 μl of 1:1000 anti-BCG rabbit serum (commercially availablefrom DAKO) preabsorbed with biotinylated TBC27 using an avidin column asdescribed in Example 2, was added and allowed to incubate at roomtemperature overnight (i.e., 18-20 hours).

The plate was washed as above, and 100 μl alkalinephosphatase-conjugated anti-rabbit Ig (commercially available fromPierce) diluted 1:5000 in BSA Blocker in TBS was added, and allowed toincubate for 2 hours with rocking as described above. The plate was thenwashed again as described above. To the washed plate, 100 μl amplifier(NADP+0.02 mM (Boehringer) in 50 mM diethanolamine (Sigma), 1 mM MgCl₂(Sigma), 0.1 mM ZnCl₂, (Sigma), and 15 mM NaN₃ (Sigma), at pH 9.5) wasadded to each well. The plate was incubated for 10 minutes at roomtemperature, during which time NAD was generated. Then, 200 μldiaphorase-dehydrogenase mixture was added (2 mg/ml alcoholdehydrogenase (Sigma), 1.5 mg/ml diaphorase (Boehringer), 4% ethanol(Sigma), 0.55 mM INT-violet (Sigma), 5 mg/ml BSA (Sigma), at pH 7.2).After 5 minutes of incubation time, the OD₄₉₂ was measured using aMaxline microplate reader (Molecular Devices). A four-parameter standardcurve relating concentration to optical density was generated usingSoftMax computer software (Molecular Devices).

Because background signal is often the limiting factor in amplifiedassays, several measures were undertaken to reduce background signal inthis assay. First, the antiserum used for detection (anti-BCG rabbitserum) of the alpha antigen was reabsorbed by passing it over astreptavidin column to which biotinylated TBC27 (ie., the primaryantibody) had been coupled. This was done to remove nonspecificreactivity against mouse IgG as well as specific alpha-anti-idiotypicantibody. Second, a conjugated anti-rabbit-Ig was selected which hadbeen depleted of reactivity with murine and human IgG. Third,biotin/avidin was used to fix the capture antibody to the plate; this inturn allowed for more vigorous washing protocols with adetergent-containing buffer. Fourth, particular blocking reagents (BSABlocker and SuperBlock, Pierce) were used, and albumin devoid ofalkaline phosphatase activity was selected (Pierce). Finally, severalsources of NADP were tested to identify reliable sources of NADP whichcontained the least amount of contaminating NAD. In multiplereproductions of this Example, it was determined that the best source ofNADP was Boehringer, whereas that from Sigma was inferior.

The results of this example are shown in FIG. 3. Data in this figure arepresented on a log-log scale to show its wide dynamic range. Thehorizontal axis reflects total protein content of M. tuberculosisculture filtrate; of this, alpha antigen represented approximately 10%,as estimated by densitometric measurement of proteinstained gels. Thus,the lower limit of detection of the assay was approximately 1 pg/ml.

EXAMPLE 4

Recovery of M. tuberculosis Alpha Antigen in Spiked Samples

In this Example, the same methods as described in Example 3 were usedfor testing of serum or urine, with the exception that M. tuberculosisculture filtrates were added to normal pooled normal human serum orurine, as described above. This experiment was conducted in order todetermine whether alpha antigen from M. tuberculosis filtrate could bedetected in such samples. The results of this experiment are shown inFIG. 4. As can be seen from FIG. 4, the sensitivity of the assay was notsubstantially affected by either diluent (i.e., normal serum or urine).However, the study suggests that separate diluents for standards beused, depending upon the type of clinical sample.

EXAMPLE 5

Radiometric Detection of M. tuberculosis Compared to Alpha AntigenDetection

Radiometric detection of growth of mycobacteria is the standard to whichclinical laboratories are held for rapid diagnosis of tuberculosis byaccrediting agencies such as the Joint Commission on Accreditation ofHealthcare Organizations (JCAHO). Thus, it was of interest to compare acommonly used radiometric method with the ELISA method of the presentinvention for the detection of M. tuberculosis.

For clinical samples of infected body fluids (e.g., sputum, etc.),standard methods of specimen processing are used, includingdecontamination and concentration. These samples are then inoculatedinto special radiometric medium which contains ¹⁴C-radiolabelledpalmitic acid. For the BACTEC system (Becton Dickinson), metabolicactivity of any organisms in the culture bottle results in production of¹⁴CO₂. The gas above the liquid medium in the bottle is sampled dailyand the ¹⁴CO₂ content measured. The result is expressed as a growthindex (GI). GI values above 20-30 are considered positive. Cultures withpositive growth indices must then be examined using other methods inorder to identify the organism(s) present.

As in the previous Example, BACTEC 14B bottles supplied by themanufacturer were inoculated with from 10⁴ to 10¹ colony forming units(CFU) of M. tuberculosis strain H37rv. Growth indices were measureddaily as recommended by the manufacturer; simultaneous samples werecollected for measurement of alpha antigen using the ELISA method of thepresent invention. On each day of measurement, 0.2 ml of medium wasremoved from the BACTEC bottle for measurement of alpha antigen, and anequal volume of fresh medium was replaced. The results of this study areshown in FIG. 5. In FIG. 5, solid data points represent growth index(GI) values, while the open data points represent alpha antigen content.The lower limit of detection of alpha antigen in this experiment was 10pg/ml. Clinical samples are generally scored as positive when growthindices reach 30. The time to positivity of the two assays is summarizedin Table 1:

TABLE 1 Comparison of BACTEC and Amplified ELISA Alpha Antigen Days toConcentration at Days to Alpha Inoculum GI > 30 GI = 30 Antigen > 10 pg10⁴  5 61 <4 10³  8 205 6 10² 12 68 8 10¹ 17 >800 13

Thus, regardless of inoculum size, growth was detected from 2 to 4 daysearlier by the antigen detection assay of the present invention, ascompared to radiometric assay.

EXAMPLE 6 Radiometric Detection of M. avium Compared to Alpha AntigenDetection

In view of the importance of M. avium, the methods described in Example5 above were used with a culture of M. avium replacing the M.tuberculosis culture used in the previous Example. This M. avium culturewas a clinical isolate obtained from the blood of an AIDS patient withdisseminated M. avium infection. This culture was propagated inProskauer Beck medium as described above, with the exception that 1%dextrose was added to the culture medium.

It was found that the amplified ELISA assay system was substantiallyless sensitive for detection of M. avium alpha antigen than for M.tuberculosis. Antigen was detected only simultaneously or within one dayof positive GI values. Despite more rapid growth of M. avium, antigenlevels were less than 10% of those found with M. tuberculosis. Table 2shows the results observed in this experiment.

TABLE 2 M. avium Detection Initial Days to Days to Alpha CFU GI > 30Antigen > 10 pg/ml 10⁴ 4 4 10³ 5 5 10² 6 6 10¹ 8 7

Nonetheless, these results indicate that the present invention may beused for the detection of alpha antigen in culture filtrates.

EXAMPLE 7 Alpha Antigen Detection in Urine and Serum of HIV-NegativeTuberculosis Patients

In this Example, urine and serum obtained from Ugandan tuberculosispatients not infected with HIV, as well as tuberculosis-negative,HIV-negative controls were tested using the amplified ELISA assaydescribed in Example 3.

These HIV-negative patients with tuberculosis had presented to theUganda TB Control Programme referral center at Mulago Hospital, Kampala,the clinical site of the Case Western Reserve University TB ResearchUnit. Pulmonary tuberculosis was diagnosed on the basis of a compatiblechest radiograph, a positive sputum AFB smear, and confirmatory growthof M. tuberculosis on culture of sputum. These patients were determinedto be HIV-1 uninfected by commercial serum ELISA test system(Cambridge). Urine and serum specimens were obtained for antigendetection assay prior to initiation of therapy for tuberculosis.

Control subjects without tuberculosis were identified from amongpatients under treatment for conditions other than tuberculosis, at thePulmonary Clinic at Mulago Hospital. Their diagnoses were asthma andchronic bronchitis. None of these patients were known or suspected to beHIV-infected. Given the prevalence of HIV infection in Kampala(approximately 8%) it is unlikely that more than one subject in thisgroup was HIV-infected. However, in view of the prevalence of PPD skintest reactivity in Kampala (approximately 70% in HIV uninfected persons;reviewed in M. Schulzer et al., “An estimate of the future size of thetuberculosis problem in sub-Saharan Africa resulting from HIVinfection,” Tuber. Lung Dis., 73:52-8 [1992], and the published erratumwhich appears in Tuber. Lung Dis., 73(4):245-6 [1992]), it is likelythat 14 of the subjects in this group had been infected with M.tuberculosis. Assuming a lifetime risk of tuberculosis of 10% in M.tuberculosis-infected HIV-negative persons, and 70% in M.tuberculosis-infected HIV-positive persons, it is likely thatapproximately one subject in this cohort will develop tuberculosis inthe future due to recrudescence of latent infection.

The amplified ELISA test system of Example 3 was used to test serum andurine of these patients. For this experiment, cutoff values of 2 ng/mland 20 pg/ml for urine and sputum, respectively, were selected. At thesevalues, the sensitivity and specificity of the assay with thesespecimens were determined. For serum samples from non-tuberculosispatients, 21 were found to be negative, while one was positive. Forserum samples from tuberculosis patients, 13 were found to be positive,while seven were found to be negative. Thus, for serum samples, thesensitivity was 35%, and the specificity was 95%. For urine samples fromnon-tuberculosis patients, 21 were found to be negative, and none werefound to be positive. For urine samples from tuberculosis patients,eight were found to be negative, while 13 were found to be positive.Thus, for urine samples, the sensitivity was 62%, and the specificitywas 100%. FIG. 6 graphically shows the results with the urine, whileFIG. 7 shows the results with the serum samples from these patients.

EXAMPLE 8 Alpha Antigen Detection in Urine of HIV-Positive TuberculosisPatients

In this study, urine samples from two cohorts of HIV infected subjectswere tested using the ELISA assay described in Example 3. The firstcohort was composed of Ugandan patients and the second was composed ofHIV patients from Cleveland, Ohio. The Ugandan HIV-infected patientswere diagnosed with tuberculosis based on the observation of atuberculosis-compatible chest radiograph, a positive sputum AFB smear,and confirmatory growth of M. tuberculosis on culture of sputum. Thesepatients were determined to be HIV-1 infected by the same commercialserum ELISA test described in Example 7.

A control Ugandan group, composed of Ugandan HIV-infected patientswithout tuberculosis was identified in the course of evaluation andenrollment in tuberculosis preventive therapy studies in Uganda. Theseindividuals had originally presented at anonymous HIV testing centers inKampala, were found to be seropositive, and were referred to the CaseWestern Reserve University tuberculosis center for possible enrollmentin preventive therapy studies. These subjects had normal chestradiographs, and had negative sputum AFB smears and cultures. The meanCD4 count of this group was 360/μl.

The second cohort included HIV-infected patients with disseminated M.avium infection identified through the Cleveland AIDS Clinic or hospitalward, on the basis of a positive blood, bone marrow, or lymph nodeculture. Three individuals were being treated for their MAC disease atthe time the urine cultures were collected,, although one individual wasincluded in this study two weeks before the diagnosis was established.Three additional individuals were studied at the time of diagnosis butprior to initiation of therapy.

A control group of HIV-infected subjects without MAC or tuberculosiswere identified at similar locations in Cleveland. Several subjects hadmultiple blood cultures for suspected M. avium disease, but none werepositive. The mean CD4 count of this group was 280/μl.

The same cutoff (2 ng/ml) was used as in Example 7. The results fordetection of urinary alpha antigen in this example are shown in FIG. 8.These results indicated that the sensitivity (77%) was increased fromthat in HIV-uninfected subjects in Example 7. Apparent false positiveswere observed in 22 of 25 HIV-infected Ugandans without tuberculosis.However, using the estimates in this group, it is likely that 18 wereactually infected with M. tuberculosis, and 12 may develop tuberculosisdue to recrudescence of latent infection. The positive values in thissubgroup may therefore reflect production of alpha antigen by tuberclebacilli contained within granulomas.

EXAMPLE 9 Detection of Alpha Antigen in Urine

Subjects for this example were identified as follows: patient withuntreated pulmonary tuberculosis were identified on the basis of apositive sputum acid fast smear with culture confirmation, and a chestradiograph compatible with pulmonary tuberculosis at the TuberculosisTreatment Centre, Kampala, Uganda. All subjects with tuberculosis weretested for HIV by ELISA. A 10% random sample of positives were confirmedby Western blot for quality assurance (using a commercial kit fromBioRad). Although CD4 cell counts were not determined in this group,recent studies performed in a similar cohort (newly diagnosed HIV+pulmonary tuberculosis) at this site found a median CD4 cell count of343/μL.

HIV-infected Ugandans without tuberculosis were enrolled through theirparticipation in a placebo control arm of a study of preventive therapyfor tuberculosis in HIV-infected persons. These subjects had chest filmsand sputum cultures at regular intervals which were negative fortuberculosis. The median CD4 cell count of this group was 355/μL.

Ugandans without tuberculosis, and who were not known to beHIV-infected, were recruited through the Chest Clinic at MulagoHospital, Kampala. This group consisted of patients with chronicbronchitis or asthma, normal chest radiographs, not known to be HIVseropositive, and never having had M. tuberculosis identified in sputum,and not known to ever have been treated with any antituberculous drugs.Based on the prevalence of HIV seroreactivity and tuberculin skin testreactivity in Kampala, approximately 15 (70%) of these subjects werelikely to PPD positive, and no more than 1-2 (10%) were likely to be HIVseropositive. Approximately half of the Ugandan population wasvaccinated once with BCG in infancy. However, tuberculin skin testreactivity in Ugandan adults is not affected by the administration ofBCG during infancy (ie., as indicated by the presence of a BCG scar);this is thought to reflect infection with M. tuberculosis.

HIV-infected persons at low risk for tuberculosis were recruited atUniversity Hospitals, Cleveland, Ohio, through the AIDS Clinical TrialsUnit. The incidence of tuberculosis among American-born persons in thegreater Cleveland area was 3.2/100,000 in 1994, one of the lowest amongmetropolitan areas in the United States. Subjects from this site werestudied if they were not known to have disseminated MAC, tuberculosis,or a history of a positive tuberculin skin test, and had not emigratedfrom a country with a high risk of tuberculosis, and had not been givenpreventive therapy for tuberculosis. The other HIV-related infectiousdiagnoses in this group included six patients with Pneumocystis cariniipneumonia (PCP), five patients with cytomegalovirus (CMV) infection,eight patients infected with herpes simplex virus or herpes zoster, fivepatients with candidiasis, one patient with toxoplasmosis, one patientwith syphilis, one patient with cryptococcosis, two patients withtracheobronchitis due to Xanthomonas or Pseudomonas, and two patientswith unspecified hepatitis. The median CD4 cell count of this patientgroup was 170/μl.

Subjects from this site identified with disseminated M. avium infection(MAC) were diagnosed on the basis of positive blood or lymph nodeculture in the setting of a compatible clinical presentation. Two of the7 subjects had simultaneous blood cultures which were positive and hadnot yet begun treatment for MAC. The median CD4 cell count of this groupwas 20/μl.

Persons at low risk for HIV infection were identified among laboratoryand university personnel. Subjects in this group who had not previouslyhad a positive tuberculin skin test, and who had not been tested withinthe past year, were skin tested tested after urine collection.Information was collected from these subjects regarding BCG vaccinationand country of origin. Subjects were excluded if they had been givenpreventive therapy for tuberculosis.

Tuberculin skin testing was performed by the Mantoux method using 5 T.U.(tuberculin units) with PPD. Induration was read at 48 hr. Results werescored as positive if induration was ≧5 or 10 mm in HIV-infected anduninfected subjects, respectively. In healthy American subjects, only a0 mm response was read as negative, and subjects with reactions of from1 to 9 mm were not included in the study.

Dual color flow cytometry was performed for enumeration of CD3+ and CD4+cells. Growth of M. tuberculosis on culture was confirmed by standardmethods, based on colonial morphology on Lowenstein-Jensen medium,nitrate and niacin positivity, and resistance to 5% thiopencarboxylicacid hydrazine.

Alpha antigen was detected by ELISA using a biotinylated monoclonalantibody TBC27 as described in Example 3. A standard curve was generatedusing serial dilutions of M. tuberculosis culture filtrate in which thealpha antigen content was determined by densitometric analysis ofcolloidal gold stained blots, in parallel with Western blot using TBC27,as described in Example 1. Pooled urine from PPD skin test-negativehealthy subjects was used as a diluent for the standard. M. tuberculosisfiltrate was prepared by precipitation in ammonium sulfate of spentProskauer Beck medium from 8 week cultures of M. tuberculosis strainH37Rv, as previously described in Example 1. M. avium filtrate wassimilarly prepared with the exception that the spent medium wassupplemented with 1% dextrose (i.e., as described in Example 6.

The relative specificity of the capture monoclonal antibody for alphaantigen of M. tuberculosis when tested by western blot is shown in FIG.9. As shown in this Figure, there is a band of reduced intensity in theM. avium lane, as compared with the M. tuberculosis lane. Representativestandard curves using culture filtrates of M. tuberculosis and M. aviumare shown in FIG. 10. In FIG. 10, the horizontal axis reflects totalprotein content of the respective mycobacterial filtrates. In M.tuberculosis filtrate, alpha antigen represents approximately 10% oftotal protein, as estimated by densitometric measurement of colloidalgold-stained blots. Thus, the threshold for detection of alpha antigenin this assay was approximately 10 pg/ml, although in some experimentsit was found to be as low as 1 pg/ml. The mid-range sensitivity of theassay for M. avium was found to be 12-fold less sensitive than that forM. tuberculosis, when the two filtrates were compared based on totalprotein content. Approximately half of this difference can be accountedby the reduced alpha antigen content of M. avium filtrate (5% instead of10% of total protein).

At high protein concentrations, the assay was found to becomeprogressively less sensitive for M. avium filtrate, approaching a100-fold difference at 1 μg/ml. In effect, this limited the maximumresponse to M. avium filtrate to the equivalent of 1-2 ng/ml M.tuberculosis alpha antigen. Additional specificity for M. tuberculosismay arise from differential secretion of proteins by the two species, inthat following infection with equal numbers of organisms inProskauer-Beck medium, growth of M. avium is more rapid, but results ina lower concentration of secreted protein.

Urinary alpha antigen levels were then evaluated in several groups ofsubjects, identified as follows, and represented in FIGS. 11 and 12. Theresults varied according to the TB and HIV status of the subjects.Log-transformed urinary antigen determinations from these subjects arepresented in these figures, so that differences between groups can bedistinguished over a wide range of values. As shown in these Figures,among HIV-seronegative subjects, antigenuria was greatest in subjectswith active untreated pulmonary tuberculosis. In this group, the medianvalue was 2.24 alpha antigen (per mg/ml). Sixty-two percent of thisgroup had values of 2 ng/ml or greater. Intermediate values wereobserved in two groups of subjects with latent infection with M.tuberculosis. These groups included Ugandan pulmonary clinic subjectswith asthma and bronchitis, and healthy PPD-positive personnel from theAmerican university cohort. None of the subjects in either group hadreceived preventive therapy for tuberculosis. The degree of antigenuriadid not differ between these two groups (median values of 79 and 50pg/ml, respectively, in Ugandan and American subjects). Urinary antigenwas undetectable in all but 2 of the skin test negative healthy Americansubjects. Only 1 of 18 subjects had antigenuria of >2 ng/ml.

Among all HIV-seronegative subjects, the median values differedsignificantly among those with active tuberculosis, those with latentinfection, and those who by skin test were uninfected, using theKruskal-Wallis analysis of variance of ranks (p<0.001). Thenon-parametric ANOVA equivalent was used because the data failed a testof normality. All three possible pair wise comparisons also differedsignificantly (p<0.05, using Dunn's method).

Antigenuria was greater in the corresponding groups of HIV-infectedsubjects. For example, in HIV-infected Ugandans with and withouttuberculosis, median urinary antigen levels were 4.3 and 8.3 ng/ml,respectively (p<0.001 for both when compared to correspondingHIV-negative groups). The median urinary alpha antigen value in AIDSpatients with disseminated MAC was 510 pg/ml; in those without MAC, itwas 79 pg/ml. Antigenuria of >2 ng/ml was observed in only one subjectwithout epidemiologic or culture evidence for M. tuberculosis infection.Among all HIV-positive subjects, the median values differed byKruskal-Wallis rank ANOVA (p<0.001). Pairwise comparisons showeddifferences between either Ugandan group and either Cleveland group(p<0.05), but not in other comparisons.

In HIV-seronegative persons, values of ≧2 ng/ml were diagnostic ofactive tuberculosis with a sensitivity of 62% and a specificity of 98%.Intermediate values (between 33 pg/ml and 2 ng/ml) were consistent witheither latent infection or active disease. These findings suggest thatthe mycobacteria in latently infected persons are not dormant, butrather are metabolically active. Thus, it is contemplated that theantigen detection assay may have potential application to distinguishthose skin test positive individuals with viable M. tuberculosis fromthose whose infection has been eradicated, either by preventive therapyor a protective immune response. This may allow for greater selectivityin application of preventive therapy for tuberculosis.

No antigen could be detected in approximately 10% of Ugandantuberculosis patients regardless of HIV status. Antibodies to otherpathogens such as HIV can be detected in urine; it is possible thatpretreatment of urine specimens to dissociate alpha antigen immunecomplexes might increase the sensitivity of the assay. Alternatively, itis possible that these patients may be infected with another member ofthe M. tuberculosis complex such as M. africanum, about which relativelylittle is known regarding alpha antigen genetics or biology.

Urinary antigen levels in HIV-infected persons were, in general, higherthan in HIV-seronegative persons, and the conclusions which can be drawnfrom test results are somewhat more limited. Values of >2 ng/ml werefound only in subjects with active tuberculosis, or with likely exposureto tuberculosis based on epidemiologic factors. This degree ofantigenuria in HIV-infected persons may be used as an indication foreither tuberculosis chemotherapy or preventive therapy, depending on theresults of additional tests. In this application, the antigen assay hada sensitivity of 84% and a specificity of 98%.

Unlike in HIV-seronegative subjects, the degree of antigenuria inHIV-infected subjects did not differ between those with latent infectionvs. active disease. The reason for this is not known. Many of theHIV-positive subjects who were studied had very high antigen levels (>10ng/ml). Thus, it is preferred that the sample contain antigen levelsthat are less than or equal to 10 ng/ml. It is contemplated that forsamples with very high antigen levels (i.e., >10 ng/ml), dilution of thesample to an antigen concentration lower than 10 ng/ml may be desirableprior to analysis. Alternatively, it is possible that in dually-infected(HIV and M. tuberculosis) persons, the total mycobacterial burden maynot be the main determinant of clinical disease, and that other factors,possibly immunologic, may be important in determining whether theinfection causes clinical illness.

Values between 33 pg and 2 ng/ml were identified in many HIV-infectedCleveland subjects who were without either active tuberculosis orepidemiologic evidence suggesting tuberculosis exposure. These levelswere significantly greater than in HIV seronegative PPD skin testnegative controls. The etiology of these results is not certain, but itmay reflect latent infection with to Mycobacterium avium. The apparentclustering of values in the range of 1-2 ng/ml in samples from thesesubjects is consistent with the behavior of the assay with M. aviumfiltrate. Disseminated infection with organisms of the Mycobacteriumavium-intracellulare complex (MAC) is the most common opportunisticinfection late in the course of HIV disease. The frequency of diseasedue to MAC rises from 3% per year for individuals with CD4 counts of100-200/μl to 39% at CD4 counts of <10/μl.

EXAMPLE 10 Detection of Alpha Antigen in Sputum

In this Example, detection of alpha antigen was studied in sputumobtained from patients at the initiation of anti-tuberculosis therapy.In this study, multiple sequential expectorated sputum samples wereobtained during the first two weeks of anti-tuberculosis therapy from agroup of HIV-uninfected AFB smear-positive subjects who presented to theCase Western Reserve University tuberculosis research unit site atVittoria, Brazil. The sputum samples were homogenized by adding 10 mgN-acetyl cysteine (Sigma) and 10 3 mm diameter glass beads (Fisher) toeach 5-10 ml sputum sample. The samples were vortexed at roomtemperature for 1 minute. Aliquots of 200 μl were frozen at −70° C.until analysis. A total of 37 pretreatment specimens was obtained from13 subjects. Using the amplified ELISA method described in Example 3,the mean alpha antigen concentration in the positive specimens wasdetermined to be 51 pg/ml. Every subject had at least one positivepre-treatment specimen for a total of 28/37 or 76% positive.

Subsequent sputum specimens were also available on days 2, 4, 7, and 14post initiation of treatment, from 10 patients. The alpha antigenconcentrations in these subsequent specimens from these subjects areshown in FIGS. 13A-15. In these Figures, the results from one patientare shown in each panel. For three patients, only the pre-treatmentspecimen was determined to positive, and all subsequent specimens werenegative. The results from this group of patients are shown in FIGS.13A-13C, with FIG. 13A showing the results for “Subject 1,”; FIG. 13B,showing the results for “Subject 10,”; and FIG. 13C showing the resultsfor “Subject 11.” In these patients, the mean initial value obtained inthe amplified ELISA was determined to be 27±13 pg/ml. The length of timealpha antigen was detectable in these specimens expressed in terms ofhalf-life was determined to be 0.6 days.

In nine of the 10 patients for which multiple sputum were tested, atleast one subsequent specimen was positive. The results for these ninepatients are shown in FIGS. 14A-14F, in panels A-F, with one patient'sresults being shown in each FIGS. 14A-14F. As can be seen from thisFigure, this group of patients tended to have higher initial antigenconcentration than the first group, although this difference did notreach statistical significance (i.e., the mean initial value was 41.4±11pg/ml). The rate of disappearance of antigen from subsequent specimensfrom this group was found to be slower than in the first group, with ahalf-life of 5.6 days.

In one patient, the level of sputum alpha antigen rose during treatment,as shown in FIG. 15. Simultaneous quantitative CFU rose more than 10fold during this interval. Quantitative CFU did not increase in any ofthe other subjects during this interval.

These results indicate that the amplified ELISA method for detection ofalpha antigen in sputum is suitable for detection of infection and/ordisease with M. tuberculosis. Importantly, these results indicate thatthis ELISA method provides significant information for monitoring theprogress of tuberculosis patients.

EXAMPLE 11 Production of Other Monoclonal Antibodies for Alpha AntigenDetection

In addition to TBC27 described and used in Example 3 above, otherantibodies were produced for potential application in the purificationand detection of alpha antigen of M. tuberculosis. These antibodies weredeveloped by immunization six week-old female Balb/c mice (CharlesRiver, Mass.) using the method of intrasplenic immunization (P. C.Svalander et al., “Intrasplenic immunization for production ofmonoclonal antibodies against mouse blastocysts,” J. Immunol. Meth.,105:221-7 [1987]). Mice were immunized with 1 μg alpha antigen obtainedfrom M. tuberculosis culture filtrate as described in Example 1. Theportion of the nitrocellulose paper containing the alpha antigen wasexcised and the paper allowed to dry at room temperature. The paper wasdissolved in DMSO (Sigma). It was then precipitated by the dropwiseaddition of sodium carbonate 0.05 M (Sigma), pH 9.6. The precipitatedparticles were then washed 3 times in 2 ml RPMI-1640 medium(BioWhittaker) and suspended in a final volume of 1 ml. The immunizationwas performed by injection into the spleen, using a 25 gauge needle.Animals were boosted with additional 1 μg twice at two week intervals,and sacrificed 2 weeks after the last boost. Spleen cells were fused toSP2/0 Ag 14 myeloma cells (ATCC) using polyethylene glycol (Sigma) andwere propagated in Dulbecco's modified Eagle' medium (Sigma) with addedhypoxanthine, aminopterin, and thymidine. Supernatants of wells withvisible growth were tested for antibody by ELISA using plates sensitizedwith M. tuberculosis filtrate as described in Example 2. Positive wellswere expanded in culture, and the supernatants collected andlyophilized, and the antibody was biotinylated using a biotinylationreagent kit (Pierce) according to the manufacturer's recommendations,all as described in Example 2.

Several antibodies specific for M. tuberculosis alpha antigen weredeveloped using the methods of this Example, including the antibodydesignated as “5C5,” and “8G104.4.” As shown in the Western blot in FIG.16, 5C5 and 8G10.4.4 reacted only with culture filtrates of M.tuberculosis and not M. smegmatis or M. avium. Thus, it is contemplatedthat these antibodies will also be useful in the amplified ELISA assayand provide increased specificity. However, with 5C5, cross-reactivitywas observed, as described in Example 12.

EXAMPLE 12 Use of 5C5 for Detection of Alpha Antigen in Urine

In this Example, antibody 5C5 produced as described in Example 11 abovewas tested for its ability to detect M. tuberculosis alpha antigen inurine. The amplified ELISA method and patient groups described inExample 7 were used, with the exception that instead of using TBC27, 5C5was used (i.e.,the samples were retested with 5C5 in place of TBC27).

The results of this study are shown in FIGS. 17 and 18. As shown in FIG.17, this antibody was capable of detecting antigen in urine. However, asshown in FIG. 18, an apparent specificity to M. tuberculosis asdetermined by Western blot (see FIG. 16) does not ensure success in thediagnosis of tuberculosis, as cross reactivity can arise. In FIG. 18,lane 1 contained M. tuberculosis, while lane 2 contained M. aviumfiltrate. As shown in FIG. 18, when biotinylated 5C5 was substituted forTBC27 in the alpha antigen ELISA, the test was of less value than theuse of TBC27 for tuberculosis diagnosis, regardless of HIV status (ie.,the results were of no value for either the HIV-positive or HIV-negativepatient groups). The spurious results were in part due to unexpectedcross reactivity of the antibody with proteins expressed on the surfaceof normal human monocytes, detected by immunohistochemistry of infectedcells.

EXAMPLE 13 Use of Synthetic Peptides to Produce Monoclonals Specific forAlpha Antigen of M. tuberculosis

In this Example, the possibility of using synthetic peptides to produceantibodies directed against M. tuberculosis alpha antigen wasinvestigated. The amino acid sequences of the alpha antigens of M. aviumand M. tuberculosis are compared in FIG. 19 (SEQ ID NOS: 1 and 2). Inthis Figure, residues which differ between the two species areemphasized in bold face. As shown in this Figure, extensive identity isapparent (87%) between the alpha antigens of these two species.

Upon inspection, potentially species-specific peptides were identifiedbeginning at amino acid residues 147 and 229 of M. tuberculosis (SEQ IDNOS: 3 and 4). SEQ ID NO: 3 is comprised of the amino acid sequence“QWLSANRAVKPTGSAAI”; while SEQ ID NO: 4 is comprised of the amino acidsequence “ERNDPTQQIPKLVAN.”

These peptides were hypothesized as representing sections that werespecific to particular species of Mycobacterium. Searches were performedusing the Entrez database search engine at the National Center forBiotechnology Information at the National Library of Medicine. Thissearch engine is based on a program developed by S. F. Altschul termed“BLASTP,” which is used to identify homologous sequences in the combinedPDB, SwissProt, PIR, GenPept, and GPupdate databases (S. F. Altschul etal., “Basic local alignment search tool,” J. Mol. Biol., 215:403-10[1990]). A facility to search these databases for homologous sequenceshas been established by the National Library of Medicine, and wasaccessed by the Internet at URALhttp://www3.ncbi.nlm.nih.gov/Entrez/index.html. The search engine allowsthe user to supply an amino acid sequence. It returns a list of allpotentially homologous sequences which have been stored in its database.The engine is designed to identify sequences which are identical, whichhave similar amino acid residues in terms of charge or size, which haveintervening inserted sequences, or which have deletions. However, in thecomparison, the “expect” search parameter was increased from a defaultof 10 to 1000 to increase the reporting of even marginal matches.

As a control, a similar search was performed for a 17 amino acidintervening sequence “DQFIYAGSLSALLDPSQ” (SEQ ID NO: 5), which begins atresidue 181 of the M. avium antigen sequence, and represents a consensusregion that is shared among the species of the Mycobacterium genus. Thissequence differs by one amino acid from the homologous stretch in the M.tuberculosis amino acid sequence. In the M. tuberculosis sequence, thefirst “D” is replaced with a “Q” (i.e., the entire sequence is“QQFIYAGSLSALLDPSQ” (SEQ ID NO: 8).

The results of these three searches are shown in the following Tables.These data were divided into four tables. The first table (Table 3)shows the comparisons for strains of mycobacteria. The second table(Table 4) shows the comparisons for various potential human pathogens.The third table (Table 5) shows the comparisons for various humanpeptides. The fourth table (Table 6) shows the comparisons for peptidesfrom other organisms.

The data in these Tables are displayed as the percent identity/homologybetween the target sequences and its best match in another species.Those matches with ≧65% identity or ≧80% homology are highlighted inbold. Items marked with an asterisk (*) required folding of at least onesequence for optimal alignment; the degree of homology of these matchesis likely overestimated. No homologous sequences were identified forthose entries left blank.

TABLE 3 Search Results for Peptides from Various Mycobacteria and ThreePeptide Sequences of Alpha Antigen of M. tuberculosis (% identity/%homology) Amino Acid Amino Acid Amino Acid 147 229 181 M. tuberculosis85-B 100/100 100/100 100/100 85-A 71/76 58/76 70/88 85-C 59/76 76/8853/65 M. bovis 85-B 100/100 100/100 100/100 Bovis antigen 71/76 58/7670/88 85-A 71/76 58/76 70/88 MPB51 53/71 M. avium 85-B 47/82 58/82100/100 M. intracellulare 58/88 70/82  94/100 Alpha Antigen M. kansasii85-B  88/100 70/88  94/100 M. scrofulaceum 64/94 82/88  94/100 AlphaAntigen M. leprae 85-A 52/88 58/76 70/76 85-B 70/83 70/76 81/93 85-C59/76 76/88 53/65

TABLE 4 Search Results for Various Human Pathogens and Three PeptideSequences of Alpha Antigen of M. tuberculosis (% identity/% homology)Amino Acid Amino Acid Amino Acid 147 229 181 Coxiella trxB, spoIIIE35/59* D-ala-D-ala Carboxypeptidase 41/76* Aspergillus P450 53 59/82* E.coli tus 35/53* Corynebacterium iron protein 35/53  Simian Paramyxovirus59/82* Bacillus cellulase 41/65* Bacillus δ endotoxin 35/71  HumanAdenovirus 53/76* Human Rotavirus A 59/59* Human Herpesvirus 29/71 Hepatitis C 29/35* Candida p450 41/65  Saccharomyces 53/82* Yeast DNAhelicase 53/82*

TABLE 5 Search Results for Human Peptides and Three Peptide Sequences ofM. tuberculosis (% identity/% homology) Amino Acid Amino Acid Amino Acid147 229 181 Tyrosine phosphatase 41/76* Testican 65/71* Isocitratedehydrogenase 35/47 T-cell receptor d Chain 29/53  TEGT (Testis enhanced 53/59* gene transcript) Granulocyte Colony 41/53 Stimulating Factor

TABLE 6 Search Results for Various Organisms and Three Peptide Sequencesof Alpha Antigen of M. tuberculosis (% identity/% homology) Amino AcidAmino Acid Amino Acid 147 229 181 Simian Herpesvirus Protein 59/76*Cladosporium gag Protein 59/59 Arabidopisis meri 5  65/71* RicePhotosystem II 35/35* Cyanophora Photosystem II 47/47* Maize PhotosystemII 29/29* Wheat Photosystem II 29/29* Shewanella Fumarate 47/59 Reductase Rubus Pyrophosphatase 59/71* Mouse Pax-1 Protein 41/41 Kluyveromyces g6p 41/51* Isomerase Cyanobacterium ATP 47/53  SynthetaseAlcaligenes CnrB 47/65  Caenorhabditis C14B9.5 53/65  41/65Synechococcus Protein 47/53  29/41 Methylophilus DCM 47/65 DehalogenaseDrosophila 41/65 47/65 Xylano Hydrolase  41/65* Curcurbita 59/59Arabidopsis  53/88* Gallus BRM Protein 35/53 Impatiens Spot Virus 41/76Phaseolus Tonoplast 41/71

EXAMPLE 14 Use of Synthetic Peptides to Produce Monoclonals Specific forAlpha Antigen of M. avium

In this example, an alternative approach to identification ofMAC-specific secreted antigens was designed based on identification ofspecies-specific epitopes of characterized antigens. The amino acidsequences of alpha antigens of M. avium and M. tuberculosis are comparedin FIG. 19 (i.e., SEQ ID NOS: 1 and 2). As discussed in Example 13,extensive identity is apparent (87%).

In this study, two candidate sequences were identified which mightconfer species specific responses to M. avium alpha antigen. The first,(SEQ ID NO: 6) with the sequence “SYLASNKGVKRTGNAAV,” begins at residue147. The second (SEQ ID NO: 7), with the sequence “QRNDPSLHIPELVGH,”begins at residue 229. To determine the potential for broad speciesspecificity in these regions, searches were performed with these aminoacid sequences using BLASTP to identify homologous sequences in thecombined PDB, SwissProt, PIR, GenPept, and GPupdate databases at theNCBI as described for the above Example. The “expect” search parameterwas increased from a default of 10 to 1000 to increase the reporting ofeven marginal matches.

As a control, a similar search was performed for a 17 amino acidintervening sequence (SEQ ID NO: 5), with the sequence“DQFIYAGSLSALLDPSQ,” which begins at residue 181 of the M. avium alphaantigen sequence, and differs by only one amino acid from the homologousM. tuberculosis sequence (SEQ ID NO: 8). As indicated above, thisstretch represents a consensus region.

The results of these three searches are shown in the following Tables.These data were divided into four tables. The first table (Table 7)shows the comparisons for strains of mycobacteria. The second table(Table 8) shows the comparisons for various potential human pathogens.The third table (Table 9) shows the comparisons for various humanpeptides. The fourth table (Table 10) shows the comparisons for peptidesfrom other organisms.

The data in these Tables are displayed as the percent identity/homologybetween the target sequences and its best match in another species.Those matches with ≧65% identity or ≧80% homology are highlighted inbold. Items marked with an asterisk (*) required folding of at least onesequence for optimal alignment; the degree of homology of these matchesis likely overestimated. No homologous sequences were identified forthose entries left blank.

TABLE 7 Search Results for Peptides from Various Mycobacteria and ThreePeptide Sequences of Alpha Antigen of M. avium (% identity/% homology)Amino Acid Amino Acid Amino Acid 147 229 181 M. avium Alpha Antigen100/100 100/100 100/100 M. intracellulare 82/88 80/86  88/100 AlphaAntigen M. bovis 85-A 47/71 46/80 71/88 85-B 47/82 94/94 85-B 47/8253/80 94/94 BCG Antigen 47/71 46/80 71/88 MPB51 47/76 53/71 MPB70/MPB80 41/53* M. kansasii 85-B 53/82 87/93 88/94 M. leprae 85-A 65/76 76/8285-B 47/76 53/66 88/94 85-C 64/76 53/73 59/71 MPT51-like Protein 53/71u1740g 47/59 M. scrofulaceum 71/76 66/93 88/94 Alpha Antigen M.tuberculosis 85-A 47/71 46/80 71/88 85-B 47/82 53/80 100/100 85-C 64/8253/73 59/71

TABLE 8 Search Results for Other Pathogens of AIDS Patients and ThreePeptide Sequences of M. avium Alpha Antigen (% identity/% homology)Amino Acid Amino Acid Amino Acid 147 229 181 Aeromonas β-lactamase 47/60Bacillus hypothetical  53/73* 49.5 KD Protein Bacillus L24 Gene Product41/47  Bacillus ORFY  53/73* Bacillus Pbp 5  53/73* Candidaβ-glucosidase 53/65 Chlamydia ItuA Gene Product 47/71* ClostridiumHypothetical 59/59* Protein Enterococcus Erythromycin 41/65* ResistanceEnterococcus Serine 41/53  Protease E. coli Glutaredoxin 3 53/71* (GRX3) E. coli pap Fimbrial 29/59  Activator Protein E. coli Hypothetical53/59* Protein f83 E. coli Pantothenate 40/53 Permease HaemophilusHeme-Binding 47/59 Protein Haemophilus HhdA 47/70  HaemophilusPreprotein 47/70 Translocase Mycoplasma genitalium  65/71* (randomgenomic) Neisseria DTDP-Glucose-46- 41/59 Dehydratase Neisseriameningitidis UDP- 41/59 Glucosyltransferase Neisseria meningitidis UDP-41/59 Glucose-4-epimeras Pseudomonas 3-methyl-2- 33/40 oxobutanoateDehydrogenase Pseudomonas Alkaline 40/60 Phosphatase H Pseudomonasputida bkdA2 33/40 Protein Saccharomyces ORF 233 41/53  Gene ProductSaccharomyces  53/67* Hypothetical Protein Ykl Saccharomyces Tubulin33/47 Suppressor Saccharomyces Ubiquitin- 47/59 activating EnzymeSaccharomyces  60/67* (Unknown Protein) Salmonella DTDP-Glucose 47/7046-Dehydratase Schizosaccharomyces 35/65 Hypothetical 26.9 kDSchizosaccharomyces 47/53 (Unknown Protein) Shigella DTDP-Glucose 47/6546-Dehydratase Shigella rfbB Protein 47/65 Streptococcus faecalis 41/65*plasmid pAM Streptococcus 41/65* agalactiae MSL Streptomyces 3-Dehydro- 40/67* quinate Dehyrogenase Streptomyces Hypothetical 47/53 Protein00929 Streptomyces 40/67 Phospho-N-acetyltransferase Treponema Flagellar35/59  Filament Core Protein Variola Major Core 53/65* Protein p4b

TABLE 9 Search Results for Human Peptides and Three Peptide Sequences ofM. avium Alpha Antigen (% identity/% homology) Amino Acid Amino AcidAmino Acid 147 229 181 Band 3 Anion Transport  53/59* Protein HistidineDecarboxylase 47/47 Ig Kappa Light Chain (VJC) 47/59* MET Gene Product41/41  Protein-Tyrosine-Phosphatase 47/60 Protein tyrosine phosphatase35/47 PTPH1 T-cell Factor 1 Splice Form F 40/47 Transforming Protein(N-myc)  60/67*

TABLE 10 Search Results for Various Organisms and Three PeptideSequences of M. avium Alpha Antigen (% identity/% homology) Amino AcidAmino Acid Amino Acid 147 228 181 Arabidopisis meri 5  53/65*Caenorhabditis F52C9.3 Gene 40/60 Product Caenorhabditis C32D5.12 53/76Gene Product Caenorhabditis 41/65 Hypothetical 53.4 KD Gene ProductCaenorhabditis LIN-9 35/65  Protein Caenorhabditis ZK637.6 35/65 Dioscorea Storage Protein  47/71* Dog Cytochrome C 53/71* DrosophilaFurin 2 53/60 Drosophila Homeobox Protein 41/53* Drosophila Kinesin-Like47/71  Protein Drosophila Maternal 47/64 Tudor Protein HalobacteriumRibosomal  47/47* Protein S8 Hordeum Lipid Transfer 59/71* Protein HorseT-cell Antigen CD2  60/80* Impatiens Virus M 41/76 Polyprotein Lamb'sQuarters  47/65* Hypothetical Protein Metalloproteinase Inhibitor 65/76*Methanosarcina vhtC  33/60* Gene Product Mouse Metalloproteinase 65/76*Inhibitor 1 Populous cellulase 43/59  Rat Dodecenoyl-CoA- 41/59* DeltaIsomerase Rat Lactogen Receptor 1 47/60 Rat Prolactin Receptor 2 47/60Rhodococcus Lipoprotein 47/59 Rice Cysteine 47/71  Proteinase InhibitorSeal Cytochrome C 53/71* Spinacia 41/70 Phosphoglucomutase StrawberryVirus 47/53 Coat Protein Styela Homeobox Protein  47/53* Stylonychia DNA47/53 Polymerase II Synechocystis 41/53  53/67 41/53 HypotheticalProtein Tobacco Superoxide 47/71* Dismutase Tomato Virus M Polyprotein41/65 Xenopus Activin Beta 53/67  41/59 B Subunit

As shown in the above Tables, the potentially species-specific sequencesgenerally established a high degree of similarity only with M.intracellulare, a member of the MAC complex, and with M. scrofulaceum,an uncommon pathogen. Little similarity was noted with proteins of otherspecies.

An additional directed search was performed to compare these same threeM. avium sequences with the PS 1 secreted antigen of the corynebacteria,which is a protein of approximately 30 kD, and which may be a member ofthe alpha antigen family (G. Joliff et al, “Cloning and nucleotidesequence of the csp1 gene encoding PS1, one of the two major secretedproteins of Corynebacterium glutamicum: the deduced N-terminal region ofPS1 is similar to the Mycobacterium antigen 85 complex,” Mol.Microbiol., 6:2349-2362 [1992]). The same methods were used in makingthese comparisons as described above. The results of this studyindicated only that there was only 24% identity and 40% homology,suggesting that cross-reactivity between the M. avium alpha antigen andthat of the corynebacteria was unlikely.

EXAMPLE 15 M. avium Alpha Antigen Peptide Sequences

Based on the analysis performed in Examples 13 and 14, peptides weresynthesized representing two potentially species-specific sequences ofM. avium alpha antigen. The first of these sequences was“SYLASNKGVKRTGNAAV” (SEQ ID NO: 6), which begins at residue 147. Thesecond sequence was “QRNDPSLHIPELVGH” (SEQ ID NO: 7), which begins atresidue 229. The peptides were synthesized on 1μ polystyrenemicroparticles by the commercial MacroMolecular Structure AnalysisFacility of the University of Kentucky, Lexington, Ky.

In the synthesis of peptides, peptides are ordinarily cleaved from thesolid support at the completion of the synthesis. However, for thepurpose of immunization, allowing the peptides to remain on the beadsmay offer several advantages. First, the particles themselves have amodest adjuvant effect, and they may be injected intrasplenicallydirectly (without use of nitrocellulose particles).

Two groups of mice as described in Example 6, above were immunized withthese peptides using both intrasplenic and conventional protocols asdescribed in Example 6 above. One animal from each group was bled totest for development of serum antibody against the peptides. Oneantibody preparation which recognized the native 30 kD alpha antigen andno other proteins in M. avium filtrate was obtained with immunizationwith the 229 peptide. Western blots of serum from these mice are shownin FIG. 20. These Westerns were prepared in the same manner as describedfor Example 1.

In addition to these preparations, it is contemplated that additionalmonoclonal and/or polyclonal antibodies be prepared that are usefull inthe present invention.

EXAMPLE 16 Comparison of the Reactivity of K-II Serum and Anti-AlphaAntigen Antibody

In this example, a series of immunoabsorption studies were conducted todemonstrate that one of the antigens recognized by the K-II (or “Kris”)serum described by Sippola et al. (Sippola et al., “Mycobacterium aviumantigenuria in patients with AIDS and disseminated M. avium disease,” J.Infect. Dis., 168:466-8 [1993]) is alpha antigen, as well as to showthat the 22.5 kD to 25 kD protein present in M. avium filtrates, is notpresent in M. tuberculosis filtrates.

To prepare the K-II serum, an adult goat was immunized by an initialinjection of an emulsion of 1 ml incomplete Freund's adjuvant (Difco) 10mg heat-killed, dried M. intracellulare serotype 5 (ATCC 35768), and 10mg dried culture filtrate of M. intracellulare serotype 5, propagatedand prepared as described above in Example 1 with glucose added to themedium. The animal was boosted twice with injection of the same quantityof M. intracellulare culture filtrate.

In this example, Western blots of culture filtrates of M. avium and M.tuberculosis were used to demonstrate the reactivity of the K-IIantiserum, and the antibodies produced in Example 2. The Western blotanalysis was performed as described above in Example 1. The K-II serumwas either used diluted 1:5000 in TBS, or was pretreated by addition of100 μg M. tuberculosis culture filtrate per 5 ml of diluted antiserum,incubated 4 hr at room temperature prior to use in Western blot.

The results of this study are shown in the Western blots of FIGS. 21(panels A-C) and 22. As shown in these blots, an M. avium-specificprotein recognized by the Kris (“K-II”) serum migrated with a molecularsize of approximately 25 kD. This results suggests a potential role forthis protein in antigen detection studies for diagnosis of MAC infectionand disease.

Several M. avium protein antigens in this molecular weight range havebeen identified and cloned on the basis of homology with other species.These include a 27 kD M. tuberculosis lipoprotein (J. Nair et al.,“Nucleotide sequence analysis and serologic characterization of a27-kilodalton Mycobacterium intracellulare lipoprotein,” Infect. Immun.,61:1074-81[1993]), and 22 and 19 kD proteins found in M. tuberculosisand M. leprae (D. P. Harris et al., “Epitope specificity and isoforms ofthe mycobacterial 19-kilodalton antigen,” Infect. Immun., 62:2963-72[1994]; R. J. Booth et al., “Homologs of Mycobacterium leprae18-kilodalton and Mycobacterium tuberculosis 19-kilodalton antigens inother mycobacteria,” Infect. Immun., 61:1509-15 [1993]; and S. L. Morriset al., “Isolation and characterization of recombinant lambda gt11bacteriophages expressing four different Mycobacterium intracellulareantigens,” Infect. Immun., 58:17-20 [1990]). However, the degree ofhomology with M. tuberculosis in these antigens make it unlikely thatany of these represent the MAC-specific Kris antigen.

EXAMPLE 17 M. avium 25 kD Protein

In this Example, the identity of the 25 kD protein identified in Example16 above is investigated in order to determine its potential role inimmunodiagnosis of MAC infection. The intraspelenic protocol describedin Example 2 is used to produce monoclonal antibodies directed againstthis protein, and hybridomas are screened for reactivity against M.avium filtrate. Those which score positive are then tested for lack ofreactivity against M. tuberculosis filtrate. Those which appear to bespecific for M. avium are expanded in culture, biotinylated, andevaluated in the antigen detection assay, as described in Example 3,with the exception that these antibodies substitute for TBC-27.

In additional experiments, K-II serum preadsorbed against the monoclonalantibody is substituted for the BCG antiserum. As K-II serum is a goatantiserum, the secondary antibody used here is an anti-goat antibody,rather than the anti-rabbit antibody used in the previous Examples.

These Examples clearly show that the amplified ELISA system of thepresent invention can be used to detect urinary mycobacterial antigen inpatients with latent and active infection. The test is potentiallyvaluable for diagnosis of tuberculosis because it can be applied tofluids remote from the site of active infection. It is rapid,inexpensive and technically straightforward, and thus has potentialapplication in less-developed regions of the world where laboratoryfacilities and fmancial resources for tuberculosis control are limited.It has the added advantage that an additional clinic visit (for skintest reading) is not required. The assay may be useful in diagnosis oflatent infection in persons in whom PPD skin testing is unreliable, asit detects microbial products rather than measuring the host response.Finally, since the assay detects a secreted (rather thancell-associated) antigen, its decline during therapy of either latent ofactive infection may be a useful, early marker of treatment success.Lastly, this is the only method currently described which canpotentially be used to monitor the effectiveness of preventive therapy.

In summary, the present invention provides numerous advances andadvantages over the prior art, including: (1) much greater sensitivity,(2) much greater safety, as there is no spillage, nor aerosolization ofbacterial cultures during biochemical testing methods; and (3) theoverall process for performing multiple tests is extremely simple andefficient, requiring very little labor on the part of themicrobiologist. All of these advantages enhance the speed and accuracyof scoring test results in studies to detect M. tuberculosis infectionand disease.

8 325 amino acids amino acid Not Relevant Not Relevant peptide 1 Met ThrAsp Val Ser Arg Lys Ile Arg Ala Trp Gly Arg Arg Leu Met 1 5 10 15 IleGly Thr Ala Ala Ala Val Val Leu Pro Gly Leu Val Gly Leu Ala 20 25 30 GlyGly Ala Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly Leu Pro Val 35 40 45 GluTyr Leu Gln Val Pro Ser Pro Ser Met Gly Arg Asp Ile Lys Val 50 55 60 GlnPhe Gln Ser Gly Gly Asn Asn Ser Pro Ala Val Tyr Leu Leu Asp 65 70 75 80Gly Leu Arg Ala Gln Asp Asp Tyr Asn Gly Trp Asp Ile Asn Thr Pro 85 90 95Ala Phe Glu Trp Tyr Tyr Gln Ser Gly Leu Ser Ile Val Met Pro Val 100 105110 Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly 115120 125 Lys Ala Gly Cys Gln Thr Tyr Lys Trp Glu Thr Phe Leu Thr Ser Glu130 135 140 Leu Pro Gln Trp Leu Ser Ala Asn Arg Ala Val Lys Pro Thr GlySer 145 150 155 160 Ala Ala Ile Gly Leu Ser Met Ala Gly Ser Ser Ala MetIle Leu Ala 165 170 175 Ala Tyr His Pro Gln Gln Phe Ile Tyr Ala Gly SerLeu Ser Ala Leu 180 185 190 Leu Asp Pro Ser Gln Gly Met Gly Pro Ser LeuIle Gly Leu Ala Met 195 200 205 Gly Asp Ala Gly Gly Tyr Lys Ala Ala AspMet Trp Gly Pro Ser Ser 210 215 220 Asp Pro Ala Trp Glu Arg Asn Asp ProThr Gln Gln Ile Pro Lys Leu 225 230 235 240 Val Ala Asn Asn Thr Arg LeuTrp Val Tyr Cys Gly Asn Gly Thr Pro 245 250 255 Asn Glu Leu Gly Gly AlaAsn Ile Pro Ala Glu Phe Leu Glu Asn Phe 260 265 270 Val Arg Ser Ser AsnLeu Lys Phe Gln Asp Ala Tyr Asn Ala Ala Gly 275 280 285 Gly His Asn AlaVal Phe Asn Phe Pro Pro Asn Gly Thr His Ser Trp 290 295 300 Glu Tyr TrpGly Ala Gln Leu Asn Ala Met Lys Gly Asp Leu Gln Ser 305 310 315 320 SerLeu Gly Ala Gly 325 330 amino acids amino acid Not Relevant Not Relevantpeptide 2 Met Thr Asp Leu Ser Glu Lys Val Arg Ala Trp Gly Arg Arg LeuLeu 1 5 10 15 Val Gly Ala Ala Ala Ala Val Thr Leu Pro Gly Leu Ile GlyLeu Ala 20 25 30 Gly Gly Ala Ala Thr Ala Asn Ala Phe Ser Arg Pro Gly LeuPro Val 35 40 45 Glu Tyr Leu Gln Val Pro Ser Ala Gly Met Gly Arg Asp IleLys Val 50 55 60 Gln Phe Gln Ser Gly Gly Asn Gly Ser Pro Ala Val Tyr LeuLeu Asp 65 70 75 80 Gly Leu Arg Ala Gln Asp Asp Tyr Asn Gly Trp Asp IleAsn Thr Pro 85 90 95 Ala Phe Glu Trp Tyr Tyr Gln Ser Gly Leu Ser Val IleMet Pro Val 100 105 110 Gly Gly Gln Ser Ser Phe Tyr Ala Asp Trp Tyr GlnPro Ala Cys Gly 115 120 125 Lys Ala Gly Cys Ser Thr Tyr Lys Trp Glu ThrPhe Leu Thr Ser Glu 130 135 140 Leu Pro Ser Tyr Leu Ala Ser Asn Lys GlyVal Lys Arg Thr Gly Asn 145 150 155 160 Ala Ala Val Gly Ile Ser Met SerGly Ser Ser Ala Met Ile Leu Ala 165 170 175 Val Asn His Pro Asp Gln PheIle Tyr Ala Gly Ser Leu Ser Ala Leu 180 185 190 Leu Asp Pro Ser Gln GlyMet Gly Pro Ser Leu Ile Gly Leu Ala Met 195 200 205 Gly Asp Ala Gly GlyTyr Lys Ala Asp Ala Met Trp Gly Pro Ser Ser 210 215 220 Asp Pro Ala TrpGln Arg Asn Asp Pro Ser Leu His Ile Pro Glu Leu 225 230 235 240 Val GlyHis Asn Thr Arg Leu Trp Leu Tyr Cys Gly Asn Gly Thr Pro 245 250 255 SerGlu Leu Gly Gly Ala Asn Met Pro Ala Glu Phe Leu Glu Asn Phe 260 265 270Val Arg Ser Ser Asn Leu Lys Phe Gln Asp Ala Tyr Asn Gly Ala Gly 275 280285 Gly His Asn Ala Val Phe Asn Phe Asn Ala Asn Gly Thr His Ser Trp 290295 300 Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys Pro Asp Leu Gln Gly305 310 315 320 Thr Leu Gly Ala Ser Pro Gly Gly Gly Gly 325 330 17 aminoacids amino acid Not Relevant Not Relevant peptide 3 Gln Trp Leu Ser AlaAsn Arg Ala Val Lys Pro Thr Gly Ser Ala Ala 1 5 10 15 Ile 15 amino acidsamino acid Not Relevant Not Relevant peptide 4 Glu Arg Asn Asp Pro ThrGln Gln Ile Pro Lys Leu Val Ala Asn 1 5 10 15 17 amino acids amino acidNot Relevant Not Relevant peptide 5 Asp Gln Phe Ile Tyr Ala Gly Ser LeuSer Ala Leu Leu Asp Pro Ser 1 5 10 15 Gln 17 amino acids amino acid NotRelevant Not Relevant peptide 6 Ser Tyr Leu Ala Ser Asn Lys Gly Val LysArg Thr Gly Asn Ala Ala 1 5 10 15 Val 15 amino acids amino acid NotRelevant Not Relevant peptide 7 Gln Arg Asn Asp Pro Ser Leu His Ile ProGlu Leu Val Gly His 1 5 10 15 17 amino acids amino acid Not Relevant NotRelevant peptide 8 Gln Gln Phe Ile Tyr Ala Gly Ser Leu Ser Ala Leu LeuAsp Pro Ser 1 5 10 15 Gln

What is claimed is:
 1. A method for identification of Mycobacteriumtuberculosis in a sample comprising: a) providing: i) a monoclonalantibody directed against an epitope on a portion of alpha antigen ofMycobacterium tuberculosis, wherein said antibody is not reactive withalpha antigen of Mycobacterium avium; ii) a sample suspected ofcontaining at least a portion of said alpha antigen of Mycobacteriumtuberculosis; and b) adding said sample to said monoclonal antibodyunder conditions such that said antibody binds to said epitope of saidportion of said alpha antigen of Mycobacterium tuberculosis, to form anantibody-antigen complex, and c) detecting said antibody-antigencomplex, under conditions such that the presence of Mycobacteriumtuberculosis is detected, wherein said detecting is selected from thegroup consisting of enzyme immunoassay, radioimmunoassay, fluorescenceimmunoassay, flocculation, particle agglutination, and in situchromogenic assay.
 2. The method of claim 1, wherein said detectingcomprises adding a primary antibody to said antigen-antibody complex sothat said primary antibody binds to said antigen to form anantibody-antigen-antibody sandwich.
 3. The method of claim 2, whereinsaid detecting further comprises adding a reporter reagent, wherein saidreporter reagent comprises an antibody reporter, to saidantibody-antigen-antibody sandwich to form anantibody-antigen-antibody-antibody sandwich.
 4. The method of claim 3,wherein said detecting further comprises adding an amplifier to saidantibody-antigen-antibody-antibody sandwich.
 5. The method of claim 1,wherein said monoclonal antibody comprises a murine monoclonal antibody.6. The method of claim 5, wherein said murine monoclonal antibody isbiotinylated.
 7. A method for identification of Mycobacteriumtuberculosis in a sample comprising: a) providing: i) a monoclonalantibody directed against an epitope on a portion of alpha antigen ofMycobacterium tuberculosis, wherein said antibody is not reactive withalpha antigen of Mycobacterium avium; ii) a sample suspected ofcontaining at least a portion of alpha antigen of Mycobacteriumtuberculosis; b) adding said sample to said monoclonal antibody underconditions such that said antibody binds to said epitope on said portionof said alpha antigen of Mycobacterium tuberculosis, in said sample toform an antibody-antigen complex; and c) detecting said antibody-antigencomplex, wherein said detecting is selected from the group consisting ofenzyme immunoassay, radioimmunoassay, fluorescence immunoassay,flocculation, particle agglutination, and in situ chromogenic assay. 8.The method of claim 7, wherein said detecting comprises adding a primaryantibody to said antigen-antibody complex so that said primary antibodybinds to said antigen to form an antibody-antigen-antibody sandwich. 9.The method of claim 8, wherein said detecting further comprises adding areporter reagent, wherein said reporter reagent comprises an antibodyreporter, to said antibody-antigen-antibody sandwich to form anantibody-antigen-antibody-antibody sandwich.
 10. The method of claim 9,wherein said detecting further comprises adding an amplifier to saidantibody-antigen-antibody-antibody sandwich.
 11. The method of claim 7,wherein said monoclonal antibody comprises a murine monoclonal antibody.12. The method of claim 11, wherein said murine monoclonal antibody isbiotinylated.